Anti-TNF Alpha Fibronectin Type III Domain Based Scaffold Compositions, Methods and Uses

ABSTRACT

A protein scaffold based on a consensus sequence of the tenth fibronectin type III (FN3) repeat from human fibronectin, preferably human Tenascin, that binds to human TNFα including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof have applications in diagnostic and/or therapeutic compositions, methods and devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/110123, filed 31 Oct. 2009, the entire contents of which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to protein scaffolds with novelproperties, including the ability to bind to cellular targets. Moreparticularly, the present invention is directed to a protein scaffoldbased on a consensus sequence of a fibronectin type III (FN3) repeat.

BACKGROUND OF THE INVENTION

Monoclonal antibodies are the most widely used class of therapeuticproteins when high affinity and specificity for a target molecule aredesired. However, non-antibody proteins that can be engineered to bindsuch targets are also of high interest in the biopharmaceuticalindustry. These “alternative scaffold” proteins may have advantages overtraditional antibodies due to their small size, lack of disulphidebonds, high stability, and ability to be expressed in prokaryotic hosts.Novel methods of purification are readily applied; they are easilyconjugated to drugs/toxins, penentrate efficiently into tissues and arereadily formatted into multispecific binders (Skerra 2000; Binz andPluckthun 2005).

One such alternative scaffold is the immunoglobulin (Ig) fold. This foldis found in the variable regions of antibodies, as well as thousands ofnon-antibody proteins. It has been shown that one such Ig protein, thetenth fibronectin type III (FN3) repeat from human fibronectin, cantolerate a number of mutations in surface exposed loops while retainingthe overall Ig-fold structure. Thus, libraries of amino acid variantshave been built into these loops and specific binders selected to anumber of different targets (Koide et al. 1998; Karatan et al. 2004).Such engineered FN3 domains have been found to bind to targets with highaffinity, while retaining important biophysical properties (Parker etal. 2005).

Desirable physical properties of potential alternative scaffoldmolecules include high thermal stability and reversibility of thermalfolding and unfolding. Several methods have been applied to increase theapparent thermal stability of proteins and enzymes, including rationaldesign based on comparison to highly similar thermostable sequences,design of stabilizing disulfide bridges, mutations to increase α-helixpropensity, engineering of salt bridges, alteration of the surfacecharge of the protein, directed evolution, and composition of consensussequences (Lehmann and Wyss 2001). High thermal stability is a desiredproperty of such scaffolds as it may increase the yield of recombinantprotein obtained, improve solubility of the purified molecule, improveactivity of intracellular scaffolds, decrease immunogenicity, andminimize the need of a cold chain in manufacturing.

SUMMARY OF THE INVENTION

The present invention provides a protein scaffold based on a fibronectintype III

(FN3) repeat protein, encoding or complementary nucleic acids, vectors,host cells, compositions, combinations, formulations, devices, andmethods of making and using them. In a preferred embodiment, the proteinscaffold is comprised of a consensus sequence of multiple FN3 domainsfrom human Tenascin-C (hereinafter “Tenascin”). In a further preferredembodiment, the protein scaffold of the present invention is a consensussequence of 15 FN3 domains. The protein scaffolds of the invention canbe designed to bind various molecules, for example, a cellular targetprotein.

The protein scaffolds of the invention may include additional moleculesor moieties, for example, the Fc region of an antibody, albumin bindingdomain, or other moiety influencing half-life. In further embodiments,the protein scaffolds of the invention may be bound to a nucleic acidmolecule that may encode the protein scaffold.

The present invention also provides at least one method for expressingat least one protein scaffold based on a consensus sequence of multipleFN3 domains, in a host cell, comprising culturing a host cell asdescribed herein under conditions wherein at least one protein scaffoldis expressed in detectable and/or recoverable amounts.

The present invention also provides at least one composition comprising(a) a protein scaffold based on a consensus sequence of multiple FN3domains and/or encoding nucleic acid as described herein; and (b) asuitable and/or pharmaceutically acceptable carrier or diluent.

The present invention further comprises a method of generating librariesof a protein scaffold based on a fibronectin type III (FN3) repeatprotein, preferably, a consensus sequence of multiple FN3 domains and,more preferably, a consensus sequence of multiple FN3 domains from humanTenascin. The library is formed by making successive generations ofscaffolds by altering (by mutation) the amino acids or the number ofamino acids in the molecules in particular positions in portions of thescaffold, e.g., loop regions. Libraries can be generated by altering theamino acid composition of a single loop or the simultaneous alterationof multiple loops. The loops that are altered can be lengthened orshortened accordingly. Such libraries can be generated to include allpossible amino acids at each position, or a designed subset of aminoacids. The library members can be used for screening by display, such asin vitro display (DNA, RNA, ribosome display) and phage display.

The protein scaffolds of the present invention provides enhancedbiophysical properties, such as stability under reducing conditions andsolubility at high concentrations; they may be expressed and folded inprokaryotic systems, such as E. coli, in eukaryotic systems, such asyeast, and in in vitro transcription/translation systems, such as therabbit reticulocyte lysate system

In an additional aspect, the present invention provides a method ofgenerating a scaffold molecule that binds to a particular target bypanning the scaffold libarary of the invention with the target anddetecting binders. In other related aspects, the invention comprisesscreening methods that may be used to generate or affinity matureprotein scaffolds with the desired activity, e.g., capable of binding totarget proteins with a certain affinity. Affinity maturation can beaccomplished by iterative rounds of mutagenesis and selection usingsystems, such as phage display or in vitro display. Mutagenesis duringthis process may be the result of site directed mutagenesis to specificscaffold residues, random mutagenesis due to error-prone PCR, DNAshuffling, and/or a combination of these techniques. The presentinvention further provides any invention described herein.

DESCRIPTION OF THE FIGURES

FIG. 1. SDS-PAGE analysis of purified Tencon performed on a NuPAGE 4-12%Bis-Tris gel (Invitrogen) and stained with coomassie blue. N stands fornative conditions and R for reduced conditions.

FIG. 2. Circular dichroism analysis of Tencon in PBS.

FIG. 3. DSC analysis of the 3^(rd) FN3 domain from Tenascin and Tenconin PBS. Melting temperatures of 54° C. and 78° C. were obtainedrespectively.

FIG. 4. Phagemid plasmid design of pTencon-pIX. Expression is driven bya Lac promoter and secretion via the OmpA signal sequence.

FIG. 5. Display of Myc-Tencon on M13 phage. ELISA results showingbinding of phage to α-Myc coated, CNTO95 coated, and uncoated wells.

FIG. 6. Loop structure of a the 3^(rd) FN3 domain of human Tenascin.

FIG. 7. Screening of output of TNFα selections by ELISA. Individualclones were tested for binding to biotinylated TNFα or biotinylated HSAas a control. (A) Represents results from library FG7 while (B)represents results from library BC6/FG7

DESCRIPTION OF THE INVENTION

The present invention provides an isolated, recombinant and/or syntheticprotein scaffold based on a fibronectin type III (FN3) repeat proteinfrom human fibronectin, including, without limitation, mammalian-derivedscaffold, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding protein scaffold basedon the tenth FN3 domain. The present invention further includes, but isnot limited to, methods of making and using such nucleic acids andprotein scaffolds, including diagnostic and therapeutic compositions,methods and devices.

The protein scaffolds of the present invention offer advantages overconventional therapeutics, such as ability to administer locally,orally, or cross the blood-brain barrier, ability to express in E. Coliallowing for increased expression of protein as a function of resourcesversus mammalian cell expression ability to be engineered intobispecific molecules that bind to multiple targets or multiple epitopesof the same target, ability to be conjugated to drugs, polymers, andprobes, ability to be formulated to high concentrations, and the abilityof such molecules to effectively penetrate diseased tissues and tumors.

Moreover, the protein scaffolds possess many of the properties ofantibodies in relation to their fold that mimics the variable region ofan antibody. This orientation enables the FN3 loops to be exposedsimilar to antibody complementarity determining regions (CDRs). Theyshould be able to bind to cellular targets and the loops can be altered,e.g., affinity matured, to improve certain binding or relatedproperties.

Three of the six loops of the protein scaffold of the inventioncorrespond topologically to the complementarity determining regions(CDRs 1-3), i.e., antigen-binding regions, of an antibody, while theremaining three loops are surface exposed in a manner similar toantibody CDRs. These loops span residues at or about positions 13-16,22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO:16 as shown in Table3 below and FIG. 6. Preferably, the loop regions at or about residues22-28, 51-54, and 75-81 are altered for binding specificity andaffinity. One or more of these loop regions are randomized with otherloop regions and/or other strands maintaining their sequence as backboneportions to populate a library and potent binders can be selected fromthe library having high affinity for a particular protein target. One ormore of the loop regions can interact with a target protein similar toan antibody CDR interaction with the protein.

The scaffolds of the present invention may incorporate other subunits,e.g., via covalent interaction. All or a portion of an antibody constantregion may be attached to the scaffold to impart antibody-likeproperties, e.g., complement activity (ADCC), half-life, etc. Forexample, effector function can be provided and/or controlled, e.g., bymodifying C1q binding and/or FcγR binding and thereby changing CDCactivity and/or ADCC activity. “Effector functions” are responsible foractivating or diminishing a biological activity (e.g., in a subject).Examples of effector functions include, but are not limited to: C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions may require the Fc region to be combined with abinding domain (e.g., protein scaffold loops) and can be assessed usingvarious assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

Additionally, a toxin conjugate, albumin or albumin binders,polyethylene glycol (PEG) molecules may be attached to the scaffoldmolecule for desired properties. Any of these fusions may be generatedby standard techniques, for example, by expression of the fusion proteinfrom a recombinant fusion gene constructed using publically availablegene sequences.

The scaffolds of the present invention can be used as monospecific inmonomeric form or as bi- or multi-specific (for different proteintargets or epitopes on the same protein target) in multimer form. Theattachments may be covalent or non-covalent. For example, a dimericbispecific scaffold has one subunit with specificity for a first targetprotein or epitope and a second subunit with specificity for a secondtarget protein or epitope. Scaffold subunits can be joined in a varietyof conformations that can increase the valency and thus the avidity ofantigen binding

As used herein, an “antibody” includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulinmolecule, such as but not limited to, at least one complementaritydetermining region (CDR) of a heavy or light chain or a ligand bindingportion thereof, a heavy chain or light chain variable region, a heavychain or light chain constant region, a framework region, or any portionthereof. Such antibody optionally further affects a specific ligand,such as but not limited to, where such antibody modulates, decreases,increases, antagonizes, agonizes, mitigates, alleviates, blocks,inhibits, abrogates and/or interferes with at least one activity orbinding, or with receptor activity or binding, in vitro, in situ and/orin vivo.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, including,without limitation, antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including, without limitation,single chain antibodies, single domain antibodies, and fragments thereofFunctional fragments include antigen-binding fragments that bind to aparticular target. For example, antibody fragments capable of binding toa particular target or portions thereof, including, but not limited to,Fab (e.g., by papain digestion), Fab′ (e.g., by pepsin digestion andpartial reduction) and F(ab′)₂ (e.g., by pepsin digestion), facb (e.g.,by plasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd(e.g., by pepsin digestion, partial reduction and reaggregation), Fv orscFv (e.g., by molecular biology techniques) fragments, are encompassedby the invention (see, e.g., Colligan, Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the CH₁ domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

A scaffold protein of the present invention can be used to measure oreffect in a cell, tissue, organ or animal (including mammals andhumans), to diagnose, monitor, modulate, treat, alleviate, help preventthe incidence of, or reduce the symptoms of, at least one disease orcondition, selected from, but not limited to, at least one of an immunedisorder or disease, a cardiovascular disorder or disease, aninfectious, malignant, and/or neurologic disorder or disease, or otherknown or specified related condition.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least onescaffold protein to a cell, tissue, organ, animal or patient in need ofsuch modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple, or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts.

Scaffold Protein of the Present Invention—Production and Generation

At least one scaffold protein of the present invention can be optionallyproduced by a cell line, a mixed cell line, an immortalized cell orclonal population of immortalized cells, as well known in the art. See,e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., MolecularCloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y.(1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold SpringHarbor, N.Y. (1989); Colligan, et al., eds., Current Protocols inImmunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al.,Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y.,(1997-2001).

Amino acids from a scaffold protein can be altered, added and/or deletedto reduce immunogenicity or reduce, enhance or modify binding, affinity,on-rate, off-rate, avidity, specificity, half-life, stability,solubility or any other suitable characteristic, as known in the art.

Optionally, scaffold proteins can be engineered with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, the scaffold proteins can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualengineered products using three-dimensional models of the parental andengineered sequences. Three-dimensional models are commonly availableand are familiar to those skilled in the art. Computer programs areavailable which illustrate and display probable three-dimensionalconformational structures of selected candidate sequences and canmeasure possible immunogenicity (e.g., Immunofilter program of Xencor,Inc. of Monrovia, Calif.). Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidatesequence, i.e., the analysis of residues that influence the ability ofthe candidate scaffold protein to bind its antigen. In this way,residues can be selected and combined from the parent and referencesequences so that the desired characteristic, such as affinity for thetarget antigen(s), is achieved. Alternatively, or in addition to, theabove procedures, other suitable methods of engineering can be used.

Screening

Screening protein scaffolds for specific binding to similar proteins orfragments can be conveniently achieved using nucleotide (DNA or RNAdisplay) or peptide display libraries, for example, in vitro display.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Thedisplayed nucleotide or peptide sequences can be from 3 to 5000 or morenucleotides or amino acids in length, frequently from 5-100 amino acidslong, and often from about 8 to 25 amino acids long. In addition todirect chemical synthetic methods for generating peptide libraries,several recombinant DNA methods have been described. One type involvesthe display of a peptide sequence on the surface of a bacteriophage orcell. Each bacteriophage or cell contains the nucleotide sequenceencoding the particular displayed peptide sequence. Such methods aredescribed in PCT Patent Publication Nos. 91/17271, 91/18980, 91/19818,and 93/08278.

Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Pat.Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, andscreening kits are commercially available from such suppliers asInvitrogen (Carlsbad, Calif.), and Cambridge Antibody Technologies(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666,4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730,5,763,733, 5,767,260, 5,856,456, assigned to Enzon; U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax, U.S. Pat.Nos. 5,427,908, 5,580,717, assigned to Affymax; U.S. Pat. No. 5,885,793,assigned to Cambridge Antibody Technologies; U.S. Pat. No. 5,750,373,assigned to Genentech, U.S. Pat. Nos. 5,618,920, 5,595,898, 5,576,195,5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan, supra;Ausubel, supra; or Sambrook, supra.

The protein scaffolds of the invention can bind human or other mammalianproteins with a wide range of affinities (K_(D)). In a preferredembodiment, at least one protein scaffold of the present invention canoptionally bind to a target protein with high affinity, for example,with a K_(D) equal to or less than about 10⁻⁷ M, such as but not limitedto, 0.1-9.9 or any range or value therein)×10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹², 10⁻¹³, 10⁻¹⁴, 10⁻¹⁵ or any range or value therein, as determinedby surface plasmon resonance or the Kinexa method, as practiced by thoseof skill in the art.

The affinity or avidity of a protein scaffold for an antigen can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al.,

“Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H.Freeman and Company: New York, N.Y. (1992); and methods describedherein). The measured affinity of a particular protein scaffold-antigeninteraction can vary if measured under different conditions (e.g., saltconcentration, pH). Thus, measurements of affinity and otherantigen-binding parameters (e.g., K_(D), K_(on), K_(off)) are preferablymade with standardized solutions of protein scaffold and antigen, and astandardized buffer, such as the buffer described herein.

Competitive assays can be performed with the protein scaffold of thepresent invention in order to determine what proteins, antibodies, andother antagonists compete for binding to a target protein with theprotein scaffold of the present invention and/or share the epitoperegion. These assays as readily known to those of ordinary skill in theart evaluate competition between antagonists or ligands for a limitednumber of binding sites on a protein. The protein and/or antibody isimmobilized or insolubilized before or after the competition and thesample bound to the target protein is separated from the unbound sample,for example, by decanting (where the protein/antibody waspreinsolubilized) or by centrifuging (where the protein/antibody wasprecipitated after the competitive reaction). Also, the competitivebinding may be determined by whether function is altered by the bindingor lack of binding of the protein scaffold to the target protein, e.g.,whether the protein scaffold molecule inhibits or potentiates theenzymatic activity of, for example, a label. ELISA and other functionalassays may be used, as well known in the art.

Nucleic Acid Molecules

Nucleic acid molecules of the present invention encoding proteinscaffolds can be in the form of RNA, such as mRNA, hnRNA, tRNA or anyother form, or in the form of DNA, including, but not limited to, cDNAand genomic DNA obtained by cloning or produced synthetically, or anycombinations thereof. The DNA can be triple-stranded, double-stranded orsingle-stranded, or any combination thereof. Any portion of at least onestrand of the DNA or RNA can be the coding strand, also known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally, with one or more introns, e.g., but not limited to, at leastone specified portion of at least one protein scaffold; nucleic acidmolecules comprising the coding sequence for a protein scaffold or loopregion that binds to the target protein; and nucleic acid moleculeswhich comprise a nucleotide sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode the protein scaffold as described herein and/or as known inthe art. Of course, the genetic code is well known in the art. Thus, itwould be routine for one skilled in the art to generate such degeneratenucleic acid variants that code for specific protein scaffolds of thepresent invention. See, e.g., Ausubel, et al., supra, and such nucleicacid variants are included in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding a protein scaffold can include,but are not limited to, those encoding the amino acid sequence of aprotein scaffold fragment, by itself; the coding sequence for the entireprotein scaffold or a portion thereof; the coding sequence for a proteinscaffold, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example, ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding a protein scaffold can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedprotein scaffold comprising an protein scaffold fragment or portion.

Polynucleotides Selectively Hybridizing to a Polynucleotide as DescribedHerein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably, at least 85% or 90% full-length sequences, and,more preferably, at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of a protein scaffold encoded by the polynucleotides describedherein. The polynucleotides of this invention embrace nucleic acidsequences that can be employed for selective hybridization to apolynucleotide encoding a protein scaffold of the present invention.See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporatedherein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, and/or (d) combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention, excluding the coding sequence, is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, are well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor, etal; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson,et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 toGyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat.No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC

Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example, a cDNA or a genomicsequence encoding a protein scaffold of the present invention, can beused to construct a recombinant expression cassette that can beintroduced into at least one desired host cell. A recombinant expressioncassette will typically comprise a polynucleotide of the presentinvention operably linked to transcriptional initiation regulatorysequences that will direct the transcription of the polynucleotide inthe intended host cell. Both heterologous and non-heterologous (i.e.,endogenous) promoters can be employed to direct expression of thenucleic acids of the present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in the intron) of a non-heterologousform of a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one protein scaffold by recombinant techniques, as is wellknown in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al.,supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but are not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one protein scaffold of the present invention can be expressedin a modified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of a protein scaffold toimprove stability and persistence in the host cell, during purification,or during subsequent handling and storage. Also, peptide moieties can beadded to a protein scaffold of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofa protein scaffold or at least one fragment thereof. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention. Alternatively, nucleic acids of thepresent invention can be expressed in a host cell by turning on (bymanipulation) in a host cell that contains endogenous DNA encoding aprotein scaffold of the present invention. Such methods are well knownin the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670,5,733,746, and 5,733,761, entirely incorporated herein by reference.

Illustrative of cell cultures useful for the production of the proteinscaffolds, specified portions or variants thereof, are bacterial, yeast,and mammalian cells as known in the art. Mammalian cell systems oftenwill be in the form of monolayers of cells although mammalian cellsuspensions or bioreactors can also be used. A number of suitable hostcell lines capable of expressing intact glycosylated proteins have beendeveloped in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7(e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g.,ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells,CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cellsand the like, which are readily available from, for example, AmericanType Culture Collection, Manassas, Va. (www.atcc.org). Preferred hostcells include cells of lymphoid origin, such as myeloma and lymphomacells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCCAccession Number CRL-1580) and SP2/0-Ag14 cells (ATCC Accession NumberCRL-1851). In a particularly preferred embodiment, the recombinant cellis a P3X63Ab8.653 or an SP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human promoter; anenhancer, and/or processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Agpoly A addition site), and transcriptional terminator sequences. See,e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells usefulfor production of nucleic acids or proteins of the present invention areknown and/or available, for instance, from the American Type CultureCollection Catalogue of Cell Lines and Hybridomas (www.atcc.org) orother known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of a Protein Scaffold

A protein scaffold can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Protein scaffolds of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, E. Coli, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the protein scaffold of the present invention canbe glycosylated or can be non-glycosylated. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Sections17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20,Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

Amino Acid Codes

The amino acids that make up protein scaffolds of the present inventionare often abbreviated. The amino acid designations can be indicated bydesignating the amino acid by its single letter code, its three lettercode, name, or three nucleotide codon(s) as is well understood in theart (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed.,Garland Publishing, Inc., New York, 1994). A protein scaffold of thepresent invention can include one or more amino acid substitutions,deletions or additions, either from natural mutations or humanmanipulation, as specified herein. Amino acids in a protein scaffold ofthe present invention that are essential for function can be identifiedby methods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15;Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity, such as, but not limited to, at least one neutralizingactivity. Sites that are critical for protein scaffold binding can alsobe identified by structural analysis, such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312(1992)).

As those of skill will appreciate, the present invention includes atleast one biologically active protein scaffold of the present invention.Biologically active protein scaffolds have a specific activity at least20%, 30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, mostpreferably, at least 80%, 90%, or 95%-1000% or more of that of thenative (non-synthetic), endogenous or related and known proteinscaffold. Methods of assaying and quantifying measures of enzymaticactivity and substrate specificity are well known to those of skill inthe art.

In another aspect, the invention relates to protein scaffolds andfragments, as described herein, which are modified by the covalentattachment of an organic moiety. Such modification can produce a proteinscaffold fragment with improved pharmacokinetic properties (e.g.,increased in vivo serum half-life). The organic moiety can be a linearor branched hydrophilic polymeric group, fatty acid group, or fatty acidester group. In particular embodiments, the hydrophilic polymeric groupcan have a molecular weight of about 800 to about 120,000 Daltons andcan be a polyalkane glycol (e.g., polyethylene glycol (PEG),polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer orpolyvinyl pyrolidone, and the fatty acid or fatty acid ester group cancomprise from about eight to about forty carbon atoms.

The modified protein scaffolds and fragments of the invention cancomprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to a protein scaffold or fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, aprotein scaffold modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying protein scaffolds of the invention can be linear or branchedand include, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theprotein scaffold of the invention has a molecular weight of about 800 toabout 150,000 Daltons as a separate molecular entity. For example,PEG₅₀₀₀ and PEG_(20,000), wherein the subscript is the average molecularweight of the polymer in Daltons, can be used. The hydrophilic polymericgroup can be substituted with one to about six alkyl, fatty acid orfatty acid ester groups. Hydrophilic polymers that are substituted witha fatty acid or fatty acid ester group can be prepared by employingsuitable methods. For example, a polymer comprising an amine group canbe coupled to a carboxylate of the fatty acid or fatty acid ester, andan activated carboxylate (e.g., activated with N,N-carbonyl diimidazole)on a fatty acid or fatty acid ester can be coupled to a hydroxyl groupon a polymer.

Fatty acids and fatty acid esters suitable for modifying proteinscaffolds of the invention can be saturated or can contain one or moreunits of unsaturation. Fatty acids that are suitable for modifyingprotein scaffolds of the invention include, for example, n-dodecanoate(C₁₂, laurate), n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈,stearate), n-eicosanoate (C₂₀, arachidate) , n-docosanoate (C₂₂,behenate), n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀),cis-A9-octadecanoate (C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate(C₂₀, arachidonate), octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like. Suitable fattyacid esters include mono-esters of dicarboxylic acids that comprise alinear or branched lower alkyl group. The lower alkyl group can comprisefrom one to about twelve, preferably, one to about six, carbon atoms.

The modified protein scaffolds and fragments can be prepared usingsuitable methods, such as by reaction with one or more modifying agents.A “modifying agent” as the term is used herein, refers to a suitableorganic group (e.g., hydrophilic polymer, a fatty acid, a fatty acidester) that comprises an activating group. An “activating group” is achemical moiety or functional group that can, under appropriateconditions, react with a second chemical group thereby forming acovalent bond between the modifying agent and the second chemical group.For example, amine-reactive activating groups include electrophilicgroups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups thatcan react with thiols include, for example, maleimide, iodoacetyl,acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol(TNB-thiol), and the like. An aldehyde functional group can be coupledto amine- or hydrazide-containing molecules, and an azide group canreact with a trivalent phosphorous group to form phosphoramidate orphosphorimide linkages. Suitable methods to introduce activating groupsinto molecules are known in the art (see for example, Hermanson, G. T.,Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Anactivating group can be bonded directly to the organic group (e.g.,hydrophilic polymer, fatty acid, fatty acid ester), or through a linkermoiety, for example, a divalent C₁-C₁₂ group wherein one or more carbonatoms can be replaced by a heteroatom, such as oxygen, nitrogen orsulfur. Suitable linker moieties include, for example, tetraethyleneglycol, —(CH₂)₃—, —NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—. Modifying agents that comprise alinker moiety can be produced, for example, by reacting amono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine,mono-Boc-diaminohexane) with a fatty acid in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amidebond between the free amine and the fatty acid carboxylate. The Bocprotecting group can be removed from the product by treatment withtrifluoroacetic acid (TFA) to expose a primary amine that can be coupledto another carboxylate, as described, or can be reacted with maleicanhydride and the resulting product cyclized to produce an activatedmaleimido derivative of the fatty acid. (See, for example, Thompson, etal., WO 92/16221, the entire teachings of which are incorporated hereinby reference.)

The modified protein scaffolds of the invention can be produced byreacting a protein scaffold or fragment with a modifying agent. Forexample, the organic moieties can be bonded to the protein scaffold in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified protein scaffolds andfragments comprising an organic moiety that is bonded to specific sitesof a protein scaffold of the present invention can be prepared usingsuitable methods, such as reverse proteolysis (Fisch et al.,Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem.,5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997);Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al.,Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described inHermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego,Calif. (1996).

Protein Scaffold Compositions Comprising Further Therapeutically ActiveIngredients

The protein scaffold compositions of the invention can optionallyfurther comprise an effective amount of at least one compound or protein(small or large molecule) selected from at least one of ananti-infective drug, a cardiovascular (CV) system drug, a centralnervous system (CNS) drug, an autonomic nervous system (ANS) drug, arespiratory tract drug, a gastrointestinal (GI) tract drug, a hormonaldrug, a drug for fluid or electrolyte balance, a hematologic drug, anantineoplastic, an immunomodulation drug, an ophthalmic, otic or nasaldrug, a topical drug, a nutritional drug or the like. Such drugs arewell known in the art, including formulations, indications, dosing andadministration for each presented herein (see, e.g., Nursing 2001Handbook of Drugs, 21^(st) edition, Springhouse Corp., Springhouse, Pa.,2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson,Stang, Prentice-Hall, Inc, Upper Saddle River, N.J.; PharmcotherapyHandbook, Wells et al., ed., Appleton & Lange, Stamford, Conn., eachentirely incorporated herein by reference).

The anti-infective drug can be at least one selected from amebicides orat least one of antiprotozoals, anthelmintics, antifungals,antimalarials, antituberculotics or at least one antileprotics,aminoglycosides, penicillins, cephalosporins, tetracyclines,sulfonamides, fluoroquinolones, antivirals, macrolide anti-infectives,and miscellaneous anti-infectives. The CV drug can be at least oneselected from inotropics, antiarrhythmics, antianginals,antihypertensives, antilipemics, and miscellaneous cardiovascular drugs.The CNS drug can be at least one selected from nonnarcotic analgesics orat least one selected from antipyretics, nonsteroidal anti-inflammatorydrugs, narcotic or at least one opiod analgesics, sedative-hypnotics,anticonvulsants, antidepressants, antianxiety drugs, antipsychotics,central nervous system stimulants, antiparkinsonians, and miscellaneouscentral nervous system drugs. The ANS drug can be at least one selectedfrom cholinergics (parasympathomimetics), anticholinergics, adrenergics(sympathomimetics), adrenergic blockers (sympatholytics), skeletalmuscle relaxants, and neuromuscular blockers. The respiratory tract drugcan be at least one selected from antihistamines, bronchodilators,expectorants or at least one antitussive, and miscellaneous respiratorydrugs. The GI tract drug can be at least one selected from antacids orat least one adsorbent or at least one antiflatulent, digestive enzymeor at least one gallstone solubilizer, antidiarrheals, laxatives,antiemetics, and antiulcer drugs. The hormonal drug can be at least oneselected from corticosteroids, androgens or at least one anabolicsteroid, estrogen or at least one progestin, gonadotropin, antidiabeticdrug or at least one glucagon, thyroid hormone, thyroid hormoneantagonist, pituitary hormone, and parathyroid-like drug. The drug forfluid and electrolyte balance can be at least one selected fromdiuretics, electrolytes or at least one replacement solution, acidifieror at least one alkalinizer. The hematologic drug can be at least oneselected from hematinics, anticoagulants, blood derivatives, andthrombolytic enzymes. The antineoplastics can be at least one selectedfrom alkylating drugs, antimetabolites, antibiotic antineoplastics,antineoplastics that alter hormone balance, and miscellaneousantineoplastics. The immunomodulation drug can be at least one selectedfrom immunosuppressants, vaccines or at least one toxoid, antitoxin orat least one antivenin, immune serum, and biological response modifier.The ophthalmic, otic, and nasal drugs can be at least one selected fromophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics,mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,otics, and nasal drugs. The topical drug can be at least one selectedfrom local anti-infectives, scabicides or at least one pediculicide ortopical corticosteroid. The nutritional drug can be at least oneselected from vitamins, minerals, or calorics. See, e.g., contents ofNursing 2001 Drug Handbook, supra.

The at least one amebicide or antiprotozoal can be at least one selectedfrom atovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, and pentamidine isethionate.The at least one anthelmintic can be at least one selected frommebendazole, pyrantel pamoate, and thiabendazole. The at least oneantifungal can be at least one selected from amphotericin B,amphotericin B cholesteryl sulfate complex, amphotericin B lipidcomplex, amphotericin B liposomal, fluconazole, flucytosine,griseofulvin microsize, griseofulvin ultramicrosize, itraconazole,ketoconazole, nystatin, and terbinafine hydrochloride. The at least oneantimalarial can be at least one selected from chloroquinehydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquinesulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine,and pyrimethamine with sulfadoxine. The at least one antituberculotic orantileprotic can be at least one selected from clofazimine, cycloserine,dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin,rifampin, rifapentine, and streptomycin sulfate. The at least oneaminoglycoside can be at least one selected from amikacin sulfate,gentamicin sulfate, neomycin sulfate, streptomycin sulfate, andtobramycin sulfate. The at least one penicillin can be at least oneselected from amoxcillin/clavulanate potassium, amoxicillin trihydrate,ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillinsodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium,mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin Gbenzathine, penicillin G potassium, penicillin G procaine, penicillin Gsodium, penicillin V potassium, piperacillin sodium, piperacillinsodium/tazobactam sodium, ticarcillin disodium, and ticarcillindisodium/clavulanate potassium. The at least one cephalosporin can be atleast one selected from cefaclor, cefadroxil, cefazolin sodium,cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium,cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetandisodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil,ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium,cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride,cephalexin monohydrate, cephradine, and loracarbef. The at least onetetracycline can be at least one selected from demeclocyclinehydrochloride, doxycycline calcium, doxycycline hyclate, doxycyclinehydrochloride, doxycycline monohydrate, minocycline hydrochloride, andtetracycline hydrochloride. The at least one sulfonamide can be at leastone selected from co-trimoxazole, sulfadiazine, sulfamethoxazole,sulfisoxazole, and sulfisoxazole acetyl. The at least onefluoroquinolone can be at least one selected from alatrofloxacinmesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacinhydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, andtrovafloxacin mesylate. The at least one fluoroquinolone can be at leastone selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin,levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,ofloxacin, sparfloxacin, and trovafloxacin mesylate. The at least oneantiviral can be at least one selected from abacavir sulfate, acyclovirsodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdinemesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium,foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine,lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivirphosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir,saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine,zanamivir, and zidovudine. The at least one macroline anti-infective canbe at least one selected from azithromycin, clarithromycin,dirithromycin, erythromycin base, erythromycin estolate, erythromycinethylsuccinate, erythromycin lactobionate, and erythromycin stearate.The at least one miscellaneous anti-infective can be at least oneselected from aztreonam, bacitracin, chloramphenicol sodium sucinate,clindamycin hydrochloride, clindamycin palmitate hydrochloride,clindamycin phosphate, imipenem and cilastatin sodium, meropenem,nitrofurantoin macrocrystals, nitrofurantoin microcrystals,quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim,and vancomycin hydrochloride. (See, e.g., pp. 24-214 of Nursing 2001Drug Handbook.)

The at least one inotropic can be at least one selected from amrinonelactate, digoxin, and milrinone lactate. The at least one antiarrhythmiccan be at least one selected from adenosine, amiodarone hydrochloride,atropine sulfate, bretylium tosylate, diltiazem hydrochloride,disopyramide, disopyramide phosphate, esmolol hydrochloride, flecainideacetate, ibutilide fumarate, lidocaine hydrochloride, mexiletinehydrochloride, moricizine hydrochloride, phenytoin, phenytoin sodium,procainamide hydrochloride, propafenone hydrochloride, propranololhydrochloride, quinidine bisulfate, quinidine gluconate, quinidinepolygalacturonate, quinidine sulfate, sotalol, tocainide hydrochloride,and verapamil hydrochloride. The at least one antianginal can be atleast one selected from amlodipidine besylate, amyl nitrite, bepridilhydrochloride, diltiazem hydrochloride, isosorbide dinitrate, isosorbidemononitrate, nadolol, nicardipine hydrochloride, nifedipine,nitroglycerin, propranolol hydrochloride, verapamil, and verapamilhydrochloride. The at least one antihypertensive can be at least oneselected from acebutolol hydrochloride, amlodipine besylate, atenolol,benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol,clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride,doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate,felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate,guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride,irbesartan, isradipine, labetalol hydrchloride, lisinopril, losartanpotassium, methyldopa, methyldopate hydrochloride, metoprolol succinate,metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol,nicardipine hydrochloride, nifedipine, nisoldipine, nitroprussidesodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate,pindolol, prazosin hydrochloride, propranolol hydrochloride, quinaprilhydrochloride, ramipril, telmisartan, terazosin hydrochloride, timololmaleate, trandolapril, valsartan, and verapamil hydrochloride. The atleast one antilipemic can be at least one selected from atorvastatincalcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride,fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin,niacin, pravastatin sodium, and simvastatin. The at least onemiscellaneous CV drug can be at least one selected from abciximab,alprostadil, arbutamine hydrochloride, cilostazol, clopidogrelbisulfate, dipyridamole, eptifibatide, midodrine hydrochloride,pentoxifylline, ticlopidine hydrochloride, and tirofiban hydrochloride.(See, e.g., pp. 215-336 of Nursing 2001 Drug Handbook.)

The at least one nonnarcotic analgesic or antipyretic can be at leastone selected from acetaminophen, aspirin, choline magnesiumtrisalicylate, diflunisal, and magnesium salicylate. The at least onenonsteroidal anti-inflammatory drug can be at least one selected fromcelecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofencalcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodiumtrihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen,naproxen sodium, oxaprozin, piroxicam, rofecoxib, and sulindac. The atleast one narcotic or opiod analgesic can be at least one selected fromalfentanil hydrochloride, buprenorphine hydrochloride, butorphanoltartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyltransdermal system, fentanyl transmucosal, hydromorphone hydrochloride,meperidine hydrochloride, methadone hydrochloride, morphinehydrochloride, morphine sulfate, morphine tartrate, nalbuphinehydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymorphonehydrochloride, pentazocine hydrochloride, pentazocine hydrochloride andnaloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride,propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate,and tramadol hydrochloride. The at least one sedative-hypnotic can be atleast one selected from chloral hydrate, estazolam, flurazepamhydrochloride, pentobarbital, pentobarbital sodium, phenobarbitalsodium, secobarbital sodium, temazepam, triazolam, zaleplon, andzolpidem tartrate. The at least one anticonvulsant can be at least oneselected from acetazolamide sodium, carbamazepine, clonazepam,clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde,fosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate,phenobarbital, phenobarbital sodium, phenytoin, phenytoin sodium,phenytoin sodium (extended), primidone, tiagabine hydrochloride,topiramate, valproate sodium, and valproic acid. The at least oneantidepressant can be at least one selected from amitriptylinehydrochloride, amitriptyline pamoate, amoxapine, bupropionhydrochloride, citalopram hydrobromide, clomipramine hydrochloride,desipramine hydrochloride, doxepin hydrochloride, fluoxetinehydrochloride, imipramine hydrochloride, imipramine pamoate,mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride,paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride,tranylcypromine sulfate, trimipramine maleate, and venlafaxinehydrochloride. The at least one antianxiety drug can be at least oneselected from alprazolam, buspirone hydrochloride, chlordiazepoxide,chlordiazepoxide hydrochloride, clorazepate dipotassium, diazepam,doxepin hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride,hydroxyzine pamoate, lorazepam, mephrobamate, midazolam hydrochloride,and oxazepam. The at least one antipsychotic drug can be at least oneselected from chlorpromazine hydrochloride, clozapine, fluphenazinedecanoate, fluephenazine enanthate, fluphenazine hydrochloride,haloperidol, haloperidol decanoate, haloperidol lactate, loxapinehydrochloride, loxapine succinate, mesoridazine besylate, molindonehydrochloride, olanzapine, perphenazine, pimozide, prochlorperazine,quetiapine fumarate, risperidone, thioridazine hydrochloride,thiothixene, thiothixene hydrochloride, and trifluoperazinehydrochloride. The at least one central nervous system stimulant can beat least one selected from amphetamine sulfate, caffeine,dextroamphetamine sulfate, doxapram hydrochloride, methamphetaminehydrochloride, methylphenidate hydrochloride, modafinil, pemoline, andphentermine hydrochloride. The at least one antiparkinsonian can be atleast one selected from amantadine hydrochloride, benztropine mesylate,biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,pramipexole dihydrochloride, ropinirole hydrochloride, selegilinehydrochloride, tolcapone, and trihexyphenidyl hydrochloride. The atleast one miscellaneous central nervous system drug can be at least oneselected from bupropion hydrochloride, donepezil hydrochloride,droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate,naratriptan hydrochloride, nicotine polacrilex, nicotine transdermalsystem, propofol, rizatriptan benzoate, sibutramine hydrochloridemonohydrate, sumatriptan succinate, tacrine hydrochloride, andzolmitriptan. (See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.)

The at least one cholinergic (e.g., parasymathomimetic) can be at leastone selected from bethanechol chloride, edrophonium chloride,neostigmine bromide, neostigmine methylsulfate, physostigminesalicylate, and pyridostigmine bromide. The at least one anticholinergiccan be at least one selected from atropine sulfate, dicyclominehydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate,propantheline bromide, scopolamine, scopolamine butylbromide, andscopolamine hydrobromide. The at least one adrenergic (sympathomimetics)can be at least one selected from dobutamine hydrochloride, dopaminehydrochloride, metaraminol bitartrate, norepinephrine bitartrate,phenylephrine hydrochloride, pseudoephedrine hydrochloride, andpseudoephedrine sulfate. The at least one adrenergic blocker(sympatholytic) can be at least one selected from dihydroergotaminemesylate, ergotamine tartrate, methysergide maleate, and propranololhydrochloride. The at least one skeletal muscle relaxant can be at leastone selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprinehydrochloride, dantrolene sodium, methocarbamol, and tizanidinehydrochloride. The at least one neuromuscular blocker can be at leastone selected from atracurium besylate, cisatracurium besylate,doxacurium chloride, mivacurium chloride, pancuronium bromide,pipecuronium bromide, rapacuronium bromide, rocuronium bromide,succinylcholine chloride, tubocurarine chloride, and vecuronium bromide.(See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)

The at least one antihistamine can be at least one selected frombrompheniramine maleate, cetirizine hydrochloride, chlorpheniraminemaleate, clemastine fumarate, cyproheptadine hydrochloride,diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine,promethazine hydrochloride, promethazine theoclate, and triprolidinehydrochloride. The at least one bronchodilator can be at least oneselected from albuterol, albuterol sulfate, aminophylline, atropinesulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate,epinephrine hydrochloride, ipratropium bromide, isoproterenol,isoproterenol hydrochloride, isoproterenol sulfate, levalbuterolhydrochloride, metaproterenol sulfate, oxtriphylline, pirbuterolacetate, salmeterol xinafoate, terbutaline sulfate, and theophylline.The at least one expectorant or antitussive can be at least one selectedfrom benzonatate, codeine phosphate, codeine sulfate, dextramethorphanhydrobromide, diphenhydramine hydrochloride, guaifenesin, andhydromorphone hydrochloride. The at least one miscellaneous respiratorydrug can be at least one selected from acetylcysteine, beclomethasonedipropionate, beractant, budesonide, calfactant, cromolyn sodium,dornase alfa, epoprostenol sodium, flunisolide, fluticasone propionate,montelukast sodium, nedocromil sodium, palivizumab, triamcinoloneacetonide, zafirlukast, and zileuton. (See, e.g., pp. 585-642 of Nursing2001 Drug Handbook.)

The at least one antacid, adsorbent, or antiflatulent can be at leastone selected from aluminum carbonate, aluminum hydroxide, calciumcarbonate, magaldrate, magnesium hydroxide, magnesium oxide,simethicone, and sodium bicarbonate. The at least one digestive enzymeor gallstone solubilizer can be at least one selected from pancreatin,pancrelipase, and ursodiol. The at least one antidiarrheal can be atleast one selected from attapulgite, bismuth subsalicylate, calciumpolycarbophil, diphenoxylate hydrochloride and atropine sulfate,loperamide, octreotide acetate, opium tincture, and opium tincure(camphorated). The at least one laxative can be at least one selectedfrom bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagradaaromatic fluidextract, cascara sagrada fluidextract, castor oil,docusate calcium, docusate sodium, glycerin, lactulose, magnesiumcitrate, magnesium hydroxide, magnesium sulfate, methylcellulose,mineral oil, polyethylene glycol or electrolyte solution, psyllium,senna, and sodium phosphates. The at least one antiemetic can be atleast one selected from chlorpromazine hydrochloride, dimenhydrinate,dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizinehydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride,perphenazine, prochlorperazine, prochlorperazine edisylate,prochlorperazine maleate, promethazine hydrochloride, scopolamine,thiethylperazine maleate, and trimethobenzamide hydrochloride. The atleast one antiulcer drug can be at least one selected from cimetidine,cimetidine hydrochloride, famotidine, lansoprazole, misoprostol,nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate,ranitidine hydrochloride, and sucralfate. (See, e.g., pp. 643-95 ofNursing 2001 Drug Handbook.)

The at least one corticosteroid can be at least one selected frombetamethasone, betamethasone acetate or betamethasone sodium phosphate,betamethasone sodium phosphate, cortisone acetate, dexamethasone,dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisoneacetate, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, prednisone,triamcinolone, triamcinolone acetonide, and triamcinolone diacetate. Theat least one androgen or anabolic steroid can be at least one selectedfrom danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate,nandrolone phenpropionate, testosterone, testosterone cypionate,testosterone enanthate, testosterone propionate, and testosteronetransdermal system. The at least one estrogen or progestin can be atleast one selected from esterified estrogens, estradiol, estradiolcypionate, estradiol/norethindrone acetate transdermal system, estradiolvalerate, estrogens (conjugated), estropipate, ethinyl estradiol,ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodioldiacetate, ethinyl estradiol and desogestrel, ethinyl estradiol andethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinylestradiol and norethindrone, ethinyl estradiol and norethindroneacetate, ethinyl estradiol and norgestimate, ethinyl estradiol andnorgestrel, ethinyl estradiol and norethindrone and acetate and ferrousfumarate, levonorgestrel, medroxyprogesterone acetate, mestranol andnorethindron, norethindrone, norethindrone acetate, norgestrel, andprogesterone. The at least one gonadroptropin can be at least oneselected from ganirelix acetate, gonadoreline acetate, histrelinacetate, and menotropins. The at least one antidiabetic or glucaon canbe at least one selected from acarbose, chlorpropamide, glimepiride,glipizide, glucagon, glyburide, insulins, metformin hydrochloride,miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazonemaleate, and troglitazone. The at least one thyroid hormone can be atleast one selected from levothyroxine sodium, liothyronine sodium,liotrix, and thyroid. The at least one thyroid hormone antagonist can beat least one selected from methimazole, potassium iodide, potassiumiodide (saturated solution), propylthiouracil, radioactive iodine(sodium iodide ¹³¹I), and strong iodine solution. The at least onepituitary hormone can be at least one selected from corticotropin,cosyntropin, desmophressin acetate, leuprolide acetate, repositorycorticotropin, somatrem, somatropin, and vasopressin. The at least oneparathyroid-like drug can be at least one selected from calcifediol,calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol,and etidronate disodium. (See, e.g., pp. 696-796 of Nursing 2001 DrugHandbook.)

The at least one diuretic can be at least one selected fromacetazolamide, acetazolamide sodium, amiloride hydrochloride,bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid,furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,spironolactone, torsemide, triamterene, and urea. The at least oneelectrolyte or replacement solution can be at least one selected fromcalcium acetate, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, calciumlactate, calcium phosphate (dibasic), calcium phosphate (tribasic),dextran (high-molecular-weight), dextran (low-molecular-weight),hetastarch, magnesium chloride, magnesium sulfate, potassium acetate,potassium bicarbonate, potassium chloride, potassium gluconate, Ringer'sinjection, Ringer's injection (lactated), and sodium chloride. The atleast one acidifier or alkalinizer can be at least one selected fromsodium bicarbonate, sodium lactate, and tromethamine. (See, e.g., pp.797-833 of Nursing 2001 Drug Handbook.)

The at least one hematinic can be at least one selected from ferrousfumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried),iron dextran, iron sorbitol, polysaccharide-iron complex, and sodiumferric gluconate complex. The at least one anticoagulant can be at leastone selected from ardeparin sodium, dalteparin sodium, danaparoidsodium, enoxaparin sodium, heparin calcium, heparin sodium, and warfarinsodium. The at least one blood derivative can be at least one selectedfrom albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitorcoagulant complex, antithrombin III (human), factor IX (human), factorIX complex, and plasma protein fractions. The at least one thrombolyticenzyme can be at least one selected from alteplase, anistreplase,reteplase (recombinant), streptokinase, and urokinase. (See, e.g., pp.834-66 of Nursing 2001 Drug Handbook.)

The at least one alkylating drug can be at least one selected frombusulfan, carboplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride,melphalan, melphalan hydrochloride, streptozocin, temozolomide, andthiotepa. The at least one antimetabolite can be at least one selectedfrom capecitabine, cladribine, cytarabine, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate,methotrexate sodium, and thioguanine The at least one antibioticantineoplastic can be at least one selected from bleomycin sulfate,dactinomycin, daunorubicin citrate liposomal, daunorubicinhydrochloride, doxorubicin hydrochloride, doxorubicin hydrochlorideliposomal, epirubicin hydrochloride, idarubicin hydrochloride,mitomycin, pentostatin, plicamycin, and valrubicin. The at least oneantineoplastic that alters hormone balance can be at least one selectedfrom anastrozole, bicalutamide, estramustine phosphate sodium,exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate,megestrol acetate, nilutamide, tamoxifen citrate, testolactone, andtoremifene citrate. The at least one miscellaneous antineoplastic can beat least one selected from asparaginase, bacillus Calmette-Guerin (BCG)(live intravesical), dacarbazine, docetaxel, etoposide, etoposidephosphate, gemcitabine hydrochloride, irinotecan hydrochloride,mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimersodium, procarbazine hydrochloride, rituximab, teniposide, topotecanhydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristinesulfate, and vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing2001 Drug Handbook.)

The at least one immunosuppressant can be at least one selected fromazathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immuneglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetilhydrochloride, sirolimus, and tacrolimus. The at least one vaccine ortoxoid can be at least one selected from BCG vaccine, cholera vaccine,diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanustoxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanustoxoids and whole-cell pertussis vaccine, Haemophilius b conjugatevaccines, hepatitis A vaccine (inactivated), hepatisis B vaccine(recombinant), influenza virus vaccine 1999-2000 trivalent types A & B(purified surface antigen), influenza virus vaccine 1999-2000 trivalenttypes A & B (subvirion or purified subvirion), influenza virus vaccine1999-2000 trivalent types A & B (whole virion), Japanese encephalitisvirus vaccine (inactivated), Lyme disease vaccine (recombinant OspA),measles and mumps and rubella virus vaccine (live), measles and mumpsand rubella virus vaccine (live attenuated), measles virus vaccine (liveattenuated), meningococcal polysaccharide vaccine, mumps virus vaccine(live), plague vaccine, pneumococcal vaccine (polyvalent), poliovirusvaccine (inactivated), poliovirus vaccine (live, oral, trivalent),rabies vaccine (adsorbed), rabies vaccine (human diploid cell), rubellaand mumps virus vaccine (live), rubella virus vaccine (live,attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoidvaccine (oral), typhoid vaccine (parenteral), typhoid Vi polysaccharidevaccine, varicella virus vaccine, and yellow fever vaccine. The at leastone antitoxin or antivenin can be at least one selected from black widowspider antivenin, Crotalidae antivenom (polyvalent), diphtheriaantitoxin (equine), and Micrurus fulvius antivenin. The at least oneimmune serum can be at least one selected from cytomegalovirus immuneglobulin (intraveneous), hepatitis B immune globulin (human), immuneglobulin intramuscular, immune globulin intravenous, rabies immuneglobulin (human), respiratory syncytial virus immune globulinintravenous (human), Rh₀(D) immune globulin (human), Rh₀(D) immuneglobulin intravenous (human), tetanus immune globulin (human), andvaricella-zoster immune globulin. The at least one biological responsemodifier can be at least one selected from aldesleukin, epoetin alfa,filgrastim, glatiramer acetate for injection, interferon alfacon-1,interferon alfa-2a (recombinant), interferon alfa-2b (recombinant),interferon beta-1a, interferon beta-1b (recombinant), interferongamma-1b, levamisole hydrochloride, oprelvekin, and sargramostim. (See,e.g., pp. 964-1040 of Nursing 2001 Drug Handbook.)

The at least one ophthalmic anti-infective can be selected formbacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin,gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamidesodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%,tobramycin, and vidarabine. The at least one ophthalmicanti-inflammatory can be at least one selected from dexamethasone,dexamethasone sodium phosphate, diclofenac sodium 0.1%, fluorometholone,flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate(suspension) and prednisolone sodium phosphate (solution). The at leastone miotic can be at least one selected from acetylocholine chloride,carbachol (intraocular), carbachol (topical), echothiophate iodide,pilocarpine, pilocarpine hydrochloride, and pilocarpine nitrate. The atleast one mydriatic can be at least one selected from atropine sulfate,cyclopentolate hydrochloride, epinephrine hydrochloride, epinephrylborate, homatropine hydrobromide, phenylephrine hydrochloride,scopolamine hydrobromide, and tropicamide. The at least one ophthalmicvasoconstrictor can be at least one selected from naphazolinehydrochloride, oxymetazoline hydrochloride, and tetrahydrozolinehydrochloride. The at least one miscellaneous ophthalmic can be at leastone selected from apraclonidine hydrochloride, betaxolol hydrochloride,brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride,dorzolamide hydrochloride, emedastine difumarate, fluorescein sodium,ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranololhydrochloride, sodium chloride (hypertonic), and timolol maleate. The atleast one otic can be at least one selected from boric acid, carbamideperoxide, chloramphenicol, and triethanolamine polypeptideoleate-condensate. The at least one nasal drug can be at least oneselected from beclomethasone dipropionate, budesonide, ephedrinesulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate,naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrinehydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide,and xylometazoline hydrochloride. (See, e.g., pp. 1041-97 of Nursing2001 Drug Handbook.)

The at least one local anti-infective can be at least one selected fromacyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazolenitrate, clindamycin phosphate, clotrimazole, econazole nitrate,erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,metronidazole (topical), miconazole nitrate, mupirocin, naftifinehydrochloride, neomycin sulfate, nitrofurazone, nystatin, silversulfadiazine, terbinafine hydrochloride, terconazole, tetracyclinehydrochloride, tioconazole, and tolnaftate. The at least one scabicideor pediculicide can be at least one selected from crotamiton, lindane,permethrin, and pyrethrins. The at least one topical corticosteroid canbe at least one selected from betamethasone dipropionate, betamethasonevalerate, clobetasol propionate, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasonepropionate, halcionide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate, andtriamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 DrugHandbook.)

The at least one vitamin or mineral can be at least one selected fromvitamin A, vitamin B complex, cyanocobalamin, folic acid,hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxinehydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D,cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol,paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodiumfluoride, sodium fluoride (topical), trace elements, chromium, copper,iodine, manganese, selenium, and zinc. The at least one caloric can beat least one selected from amino acid infusions (crystalline), aminoacid infusions in dextrose, amino acid infusions with electrolytes,amino acid infusions with electrolytes in dextrose, amino acid infusionsfor hepatic failure, amino acid infusions for high metabolic stress,amino acid infusions for renal failure, dextrose, fat emulsions, andmedium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 DrugHandbook.)

Protein scaffold compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising a protein scaffoldcontacted or administered to a cell, tissue, organ, animal or patient inneed of such modulation, treatment or therapy, optionally furthercomprising at least one selected from at least one TNF antagonist (e.g.,but not limited to a TNF chemical or protein antagonist, TNF monoclonalor polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55,p70 or p85) or fragment, fusion polypeptides thereof, or a smallmolecule TNF antagonist, e.g., TNF binding protein I or II (TBP-1 orTBP-II), nerelimonmab, infliximab, etanercept, CDP-571, CDP-870,afelimomab, lenercept, and the like), an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Non-limiting examples of such cytokines include,but are not limted to, any of IL-1 to IL-28 (e.g., IL-1, IL-2, etc.).Suitable dosages are well known in the art. See, e.g., Wells et al.,eds., Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),each of which references are entirely incorporated herein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one protein scaffold of the present invention. The toxin canoptionally act to selectively kill the pathologic cell or tissue. Thepathologic cell can be a cancer or other cell. Such toxins can be, butare not limited to, purified or recombinant toxin or toxin fragmentcomprising at least one functional cytotoxic domain of toxin, e.g.,selected from at least one of ricin, diphtheria toxin, a venom toxin, ora bacterial toxin. The term toxin also includes both endotoxins andexotoxins produced by any naturally occurring, mutant or recombinantbacteria or viruses which may cause any pathological condition in humansand other mammals, including toxin shock, which can result in death.Such toxins may include, but are not limited to, enterotoxigenic E. coliheat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigellacytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1(TSST-1), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC),Streptococcal enterotoxins and the like. Such bacteria include, but arenot limited to, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholerasuis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium dificile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophile, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Protein scaffold compounds, compositions or combinations of the presentinvention can further comprise at least one of any suitable auxiliary,such as, but not limited to, diluent, binder, stabilizer, buffers,salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the protein scaffold, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include, but are not limited to, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars, such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and the like. Representative amino acid/protein components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like. One preferred amino acid isglycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Protein scaffold compositions can also include a buffer or apH-adjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid salts,such as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts, such as citrate.

Additionally, protein scaffold compositions of the invention can includepolymeric excipients/additives, such as polyvinylpyrrolidones, ficolls(a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the protein scaffold, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferrredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents. An exemplarycarrier molecule is the mucopolysaccharide, hyaluronic acid, which maybe useful for intraarticular delivery.

Formulations

As noted above, the invention provides for stable formulations, whichpreferably comprise a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservativeas well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one protein scaffold in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,polymers, or mixtures thereof in an aqueous diluent. Any suitableconcentration or mixture can be used as known in the art, such as about0.0015%, or any range, value, or fraction therein. Non-limiting examplesinclude, no preservative, about 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4,0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5,1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0%alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075,0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9,1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one protein scaffold with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one protein scaffold, and asecond vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one protein scaffold inthe aqueous diluent to form a solution that can be held over a period oftwenty-four hours or greater.

The at least one protein scaffold used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one protein scaffold in the product of the presentinvention includes amounts yielding upon reconstitution, if in a wet/drysystem, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g., isotonicity agents, buffers, antioxidants, andpreservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably, the formulations of the presentinvention have a pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably, sodium phosphate,particularly, phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators, such asEDTA and EGTA, can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one protein scaffold and a preservativeselected from the group consisting of phenol, m-cresol, p-cresol,o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one protein scaffold andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one protein scaffold inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one proteinscaffold that is reconstituted with a second vial containing water, apreservative and/or excipients, preferably, a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period ranging from immediate to twenty-four hoursor greater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient.

Formulations of the invention can optionally be safely stored attemperatures of from about 2° C. to about 40° C. and retain thebiological activity of the protein for extended periods of time, thusallowing a package label indicating that the solution can be held and/orused over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.If preserved diluent is used, such label can include use up to 1-12months, one-half, one and a half, and/or two years.

The solutions of at least one protein scaffold of the invention can beprepared by a process that comprises mixing at least one proteinscaffold in an aqueous diluent. Mixing is carried out using conventionaldissolution and mixing procedures. To prepare a suitable diluent, forexample, a measured amount of at least one protein scaffold in water orbuffer is combined in quantities sufficient to provide the protein and,optionally, a preservative or buffer at the desired concentrations.Variations of this process would be recognized by one of ordinary skillin the art. For example, the order the components are added, whetheradditional additives are used, the temperature and pH at which theformulation is prepared, are all factors that can be optimized for theconcentration and means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one proteinscaffold that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneprotein scaffold that is reconstituted with a second vial containing theaqueous diluent. The clear solution in this case can be up to one literor even larger in size, providing a large reservoir from which smallerportions of the at least one protein scaffold solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising single vial systems include pen-injectordevices for delivery of a solution, such as BD Pens, BD Autojector®,Humaject®^(,) NovoPen®, B-D®Pen, AutoPen®, and OptiPen®, GenotropinPen®,Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®,Iject®, J-tip Needle-Free Injector®, Intrject®, Medi-Ject®, e.g., asmade or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com), and similary suitable devices. Recognized devicescomprising a dual vial system include those pen-injector systems forreconstituting a lyophilized drug in a cartridge for delivery of thereconstituted solution, such as the HumatroPen®. Examples of otherdevices suitable include pre-filled syringes, auto-injectors, needlefree injectors and needle free IV infusion sets.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute at least one protein scaffold in the aqueousdiluent to form a solution and to use the solution over a period of 2-24hours or greater for the two vial, wet/dry, product. For the singlevial, solution product, the label indicates that such solution can beused over a period of 2-24 hours or greater. The presently claimedproducts are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one protein scaffold and a selectedbuffer, preferably, a phosphate buffer containing saline or a chosensalt. Mixing at least one protein scaffold and buffer in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one protein scaffold in water or buffer is combinedwith the desired buffering agent in water in quantities sufficient toprovide the protein and buffer at the desired concentrations. Variationsof this process would be recognized by one of ordinary skill in the art.For example, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilizedprotein scaffold that is reconstituted with a second vial containing apreservative or buffer and excipients in an aqueous diluent. Either asingle solution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus provides a more convenient treatment regimenthan currently available.

Other formulations or methods of stabilizing the protein scaffold mayresult in other than a clear solution of lyophilized powder comprisingthe protein scaffold. Among non-clear solutions are formulationscomprising particulate suspensions, said particulates being acomposition containing the protein scaffold in a structure of variabledimension and known variously as a microsphere, microparticle,nanoparticle, nanosphere, or liposome. Such relatively homogenous,essentially spherical, particulate formulations containing an activeagent can be formed by contacting an aqueous phase containing the activeagent and a polymer and a nonaqueous phase followed by evaporation ofthe nonaqueous phase to cause the coalescence of particles from theaqueous phase as taught in U.S. Pat. No. 4,589,330. Porousmicroparticles can be prepared using a first phase containing activeagent and a polymer dispersed in a continuous solvent and removing saidsolvent from the suspension by freeze-drying ordilution-extraction-precipitation as taught in U.S. Pat. No. 4,818,542.Preferred polymers for such preparations are natural or syntheticcopolymers or polymers selected from the group consisting of gleatinagar, starch, arabinogalactan, albumin, collagen, polyglycolic acid,polylactic aced, glycolide-L(−) lactide poly(episilon-caprolactone,poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon-caprolactone-CO-glycolic acid), poly(B-hydroxy butyricacid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate),poly(hydroxyethyl methacrylate), polyamides, poly(amino acids),poly(2-hydroxyethyl DL-aspartamide), poly(ester urea),poly(L-phenylalanine/ethylene glycol/1,6-diisocyanatohexane) andpoly(methyl methacrylate).

Particularly preferred polymers are polyesters, such as polyglycolicacid, polylactic aced, glycolide-L(−) lactidepoly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid),and poly(epsilon-caprolactone-CO-glycolic acid. Solvents useful fordissolving the polymer and/or the active include: water,hexafluoroisopropanol, methylenechloride, tetrahydrofuran, hexane,benzene, or hexafluoroacetone sesquihydrate. The process of dispersingthe active containing phase with a second phase may include pressureforcing said first phase through an orifice in a nozzle to affectdroplet formation.

Dry powder formulations may result from processes other thanlyophilization, such as by spray drying or solvent extraction byevaporation or by precipitation of a crystalline composition followed byone or more steps to remove aqueous or nonaqueous solvent. Preparationof a spray-dried protein scaffold preparation is taught in U.S. Pat. No.6,019,968. The protein scaffold-based dry powder compositions may beproduced by spray drying solutions or slurries of the protein scaffoldand, optionally, excipients, in a solvent under conditions to provide arespirable dry powder. Solvents may include polar compounds, such aswater and ethanol, which may be readily dried. Protein scaffoldstability may be enhanced by performing the spray drying procedures inthe absence of oxygen, such as under a nitrogen blanket or by usingnitrogen as the drying gas. Another relatively dry formulation is adispersion of a plurality of perforated microstructures dispersed in asuspension medium that typically comprises a hydrofluoroalkanepropellant as taught in WO 9916419. The stabilized dispersions may beadministered to the lung of a patient using a metered dose inhaler.Equipment useful in the commercial manufacture of spray driedmedicaments are manufactured by Buchi Ltd. or Niro Corp.

At least one protein scaffold in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications

The present invention also provides a method for modulating or treatinga disease, in a cell, tissue, organ, animal, or patient, as known in theart or as described herein, using at least one protein scaffold of thepresent invention, e.g., administering or contacting the cell, tissue,organ, animal, or patient with a therapeutic effective amount of proteinscaffold. The present invention also provides a method for modulating ortreating a disease, in a cell, tissue, organ, animal, or patientincluding, but not limited to, at least one of obesity, an immunerelated disease, a cardiovascular disease, an infectious disease, amalignant disease or a neurologic disease.

The present invention also provides a method for modulating or treatingat least one immune related disease, in a cell, tissue, organ, animal,or patient including, but not limited to, at least one of rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,gastric ulcer, seronegative arthropathies, osteoarthritis, osteolysis,aseptic loosening of orthopedic implants, inflammatory bowel disease,ulcerative colitis, systemic lupus erythematosus, antiphospholipidsyndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonaryfibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis,orchitis/vasectomy reversal procedures, allergic/atopic diseases,asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergicconjunctivitis, hypersensitivity pneumonitis, transplants, organtransplant rejection, graft-versus-host disease, systemic inflammatoryresponse syndrome, sepsis syndrome, gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiationexposure, acute pancreatitis, adult respiratory distress syndrome,rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatorypathologies, sarcoidosis, Crohn's pathology, sickle cell anemia,diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergicrhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis,asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,hemolytic disesease, thrombocytopenia, graft rejection of any organ ortissue, kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynaud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, POEMS syndrome (polyneuropathy,organomegaly, endocrinopathy, monoclonal gammopathy, and skin changessyndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonalgammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus,scleroderma, mixed connective tissue disease, idiopathic Addison'sdisease, diabetes mellitus, chronic active hepatitis, primary billiarycirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IVhypersensitivity, contact dermatitis, hypersensitivity pneumonitis,allograft rejection, granulomas due to intracellular organisms, drugsensitivity, metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-l-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to, asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g., theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al.,eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, acute coronary syndrome, arteriosclerosis,atherosclerosis, restenosis, diabetic ateriosclerotic disease,hypertension, arterial hypertension, renovascular hypertension, syncope,shock, syphilis of the cardiovascular system, heart failure, corpulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrialectopic beats, atrial flutter, atrial fibrillation (sustained orparoxysmal), post perfusion syndrome, cardiopulmonary bypassinflammation response, chaotic or multifocal atrial tachycardia, regularnarrow QRS tachycardia, specific arrythmias, ventricular fibrillation,His bundle arrythmias, atrioventricular block, bundle branch block,myocardial ischemic disorders, coronary artery disease, angina pectoris,myocardial infarction, cardiomyopathy, dilated congestivecardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,endocarditis, pericardial disease, cardiac tumors, aordic and peripheralaneuryisms, aortic dissection, inflammation of the aorta, occlusion ofthe abdominal aorta and its branches, peripheral vascular disorders,occlusive arterial disorders, peripheral atherlosclerotic disease,thromboangitis obliterans, functional peripheral arterial disorders,Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venousdiseases, venous thrombosis, varicose veins, arteriovenous fistula,lymphederma, lipedema, unstable angina, reperfusion injury, post pumpsyndrome, ischemia-reperfusion injury, and the like. Such a method canoptionally comprise administering an effective amount of a compositionor pharmaceutical composition comprising at least one protein scaffoldto a cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

The present invention also provides a method for modulating or treatingat least infectious disease in a cell, tissue, organ, animal or patient,including, but not limited to, at least one of: acute or chronicbacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (e.g., A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, viral-associated hemaphagocytic syndrome, viralencephalitis/aseptic meningitis, and the like.

The present invention also provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: leukemia, acuteleukemia, acute lymphoblastic leukemia (ALL), acute lymphocyticleukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acutemyelogenous leukemia, chromic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome(MDS), a lymphoma, Hodgkin's disease, a malignamt lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, bladder cancer,breast cancer, colorectal cancer, endometiral cancer, head cancer, neckcancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, livercancer, lung cancer, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, testicularcancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like.

The present invention also provides a method for modulating or treatingat least one neurologic disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders, suchas lesions of the corticospinal system; disorders of the basal ganglia;hyperkinetic movement disorders, such as Huntington's Chorea and senilechorea; drug-induced movement disorders, such as those induced by drugswhich block CNS dopamine receptors; hypokinetic movement disorders, suchas Parkinson's disease; Progressive supranucleo Palsy; structurallesions of the cerebellum; spinocerebellar degenerations, such as spinalataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiplesystems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, andMachado-Joseph); systemic disorders (Refsum's disease,abetalipoprotemia, ataxia, telangiectasia, and mitochondrialmulti-system disorder); demyelinating core disorders, such as multiplesclerosis, acute transverse myelitis; and disorders of the motor unit,such as neurogenic muscular atrophies (anterior horn cell degeneration,such as amyotrophic lateral sclerosis, infantile spinal muscular atrophyand juvenile spinal muscular atrophy); Alzheimer's disease; Down'sSyndrome in middle age; Diffuse Lewy body disease; Senile Dementia ofLewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism;Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis,Hallerrorden-Spatz disease; Dementia pugilistica; neurotraumatic injury(e.g., spinal cord injury, brain injury, concussion, repetitiveconcussion); pain; inflammatory pain; autism; depression; stroke;cognitive disorders; epilepsy; and the like. Such a method canoptionally comprise administering an effective amount of a compositionor pharmaceutical composition comprising at least one TNF antibody orspecified portion or variant to a cell, tissue, organ, animal or patientin need of such modulation, treatment or therapy. See, e.g., the MerckManual, 16^(th) Edition, Merck & Company, Rahway, N.J. (1992).

The present invention also provides a method for modulating or treatingat least one wound, trauma or tissue injury or related chroniccondition, in a cell, tissue, organ, animal or patient, including, butnot limited to, at least one of: bodily injury or a trauma associatedwith oral surgery including periodontal surgery, tooth extraction(s),endodontic treatment, insertion of tooth implants, application and useof tooth prosthesis; or wherein the wound is selected from the groupconsisting of aseptic wounds, contused wounds, incised wounds, laceratedwounds, non-penetrating wounds, open wounds, penetrating wounds,perforating wounds, puncture wounds, septic wounds, infarctions andsubcutaneous wounds; or wherein the wound is selected from the groupconsisting of ischemic ulcers, pressure sores, fistulae, severe bites,thermal burns and donor site wounds; or wherein the wound is an aphthouswound, a traumatic wound or a herpes associated wound.

Wounds and/or ulcers are normally found protruding from the skin or on amucosal surface or as a result of an infarction in an organ (“stroke”).A wound may be a result of a soft tissue defect or a lesion or of anunderlying condition. In the present context, the term “skin” relates tothe outermost surface of the body of an animal, including a human, andembraces intact or almost intact skin as well as an injured skinsurface. The term “mucosa” relates to undamaged or damaged mucosa of ananimal, such as a human, and may be the oral, buccal, aural, nasal,lung, eye, gastrointestinal, vaginal, or rectal mucosa.

In the present context the term “wound” denotes a bodily injury withdisruption of the normal integrity of tissue structures. The term isalso intended to encompass the terms “sore,” “lesion,” “necrosis,” and“ulcer.” Normally, the term “sore” is a popular term for almost anylesion of the skin or mucous membranes and the term “ulcer” is a localdefect, or excavation, of the surface of an organ or tissue, which isproduced by the sloughing of necrotic tissue. Lesion generally relatesto any tissue defect. Necrosis is related to dead tissue resulting frominfection, injury, inflammation or infarctions.

The term “wound” used in the present context denotes any wound (seebelow for a classification of wounds) and at any particular stage in thehealing process, including the stage before any healing has initiated oreven before a specific wound like a surgical incision is made(prophylactic treatment). Examples of wounds which can be preventedand/or treated in accordance with the present invention are, e.g.,aseptic wounds, contused wounds, incised wounds, lacerated wounds,non-penetrating wounds (i.e., wounds in which there is no disruption ofthe skin but there is injury to underlying structures), open wounds,penetrating wounds, perforating wounds, puncture wounds, septic wounds,subcutaneous wounds, etc. Examples of sores are bed sores, canker sores,chrome sores, cold sores, pressure sores, etc. Examples of ulcers are,e.g., a peptic ulcer, duodenal ulcer, gastric ulcer, gouty ulcer,diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris(venous ulcer), sublingual ulcer, submucous ulcer, symptomatic ulcer,trophic ulcer, tropical ulcer, and veneral ulcer, e.g., caused bygonorrhoea (including urethritis, endocervicitis and proctitis).Conditions related to wounds or sores which may be successfully treatedaccording to the invention are burns, anthrax, tetanus, gas gangrene,scarlatina, erysipelas, sycosis barbae, folliculitis, impetigocontagiosa, or impetigo bullosa, etc. There is often a certain overlapbetween the use of the terms “wound” and “ulcer” and “wound” and “sore”and, furthermore, the terms are often used at random. Therefore, asmentioned above, in the present context the term “wound” encompasses theterms “ulcer,” “lesion,” “sore” and “infarction,” and the terms areindiscriminately used unless otherwise indicated.

The kinds of wounds to be treated according to the invention includealso (i) general wounds, such as, e.g., surgical, traumatic, infectious,ischemic, thermal, chemical and bullous wounds; (ii) wounds specific forthe oral cavity, such as, e.g., post-extraction wounds, endodonticwounds especially in connection with treatment of cysts and abscesses,ulcers and lesions of bacterial, viral or autoimmunological origin,mechanical, chemical, thermal, infectious and lichenoid wounds; herpesulcers, stomatitis aphthosa, acute necrotising ulcerative gingivitis andburning mouth syndrome are specific examples; and (iii) wounds on theskin, such as, e.g., neoplasm, burns (e.g. chemical, thermal), lesions(bacterial, viral, autoimmunological), bites and surgical incisions.Another way of classifying wounds is as (i) small tissue loss due tosurgical incisions, minor abrasions and minor bites, or as (ii)significant tissue loss. The latter group includes ischemic ulcers,pressure sores, fistulae, lacerations, severe bites, thermal burns anddonor site wounds (in soft and hard tissues) and infarctions.

Other wounds that are of importance in connection with the presentinvention are wounds like ischemic ulcers, pressure sores, fistulae,severe bites, thermal burns and donor site wounds. Ischemic ulcers andpressure sores are wounds which normally only heal very slowly andespecially in such cases, an improved and more rapid healing process isof course of great importance for the patient. Furthermore, the costsinvolved in the treatment of patients suffering from such wounds aremarkedly reduced when the healing is improved and takes place morerapidly.

Donor site wounds are wounds which, e.g., occur in connection withremoval of hard tissue from one part of the body to another part of thebody, e.g., in connection with transplantation. The wounds resultingfrom such operations are very painful and an improved healing istherefore most valuable. The term “skin” is used in a very broad senseembracing the epidermal layer of the skin and—in those cases where theskin surface is more or less injured—also the dermal layer of the skin.Apart from the stratum corneum, the epidermal layer of the skin is theouter (epithelial) layer and the deeper connective tissue layer of theskin is called the dermis.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one protein scaffold to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such diseases or disorders, wherein theadministering of said at least one protein scaffold, specified portionor variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to, a TNF chemical or proteinantagonist, TNF monoclonal or polyclonal antibody or fragment, a solubleTNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptidesthereof, or a small molecule TNF antagonist, e.g., TNF binding protein Ior II (TBP-1 or TBP-II), nerelimonmab, infliximab, etanercept (Enbrel™),adalimulab (Humira™), CDP-571, CDP-870, afelimomab, lenercept, and thelike), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, gold sodium thiomalate, hydroxychloroquine sulfate,leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21^(st) edition,Springhouse Corp., Springhouse, PA, 2001; Health Professional's DrugGuide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, UpperSaddle River, N.J. each of which references are entirely incorporatedherein by reference.

Cytokines include any known cytokine See, e.g., CopewithCytokines.com.Cytokine antagonists include, but are not limited to, any proteinscaffold, antibody, fragment or mimetic, any soluble receptor, fragmentor mimetic, any small molecule antagonist, or any combination thereof.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least protein scaffoldcomposition that total, on average, a range from at least about 0.01 to500 milligrams of at least one protein scaffold per kilogram of patientper dose, and, preferably, from at least about 0.1 to 100 milligramsprotein scaffold/kilogram of patient per single or multipleadministration, depending upon the specific activity of the active agentcontained in the composition. Alternatively, the effective serumconcentration can comprise 0.1-5000 μg/ml serum concentration per singleor multiple adminstration. Suitable dosages are known to medicalpractitioners and will, of course, depend upon the particular diseasestate, specific activity of the composition being administered, and theparticular patient undergoing treatment. In some instances, to achievethe desired therapeutic amount, it can be necessary to provide forrepeated administration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include about 0.1-99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of about 0.1-5000 μg/ml serumconcentration per single or multiple administration, or any range, valueor fraction thereof. A preferred dosage range for the protein scaffoldof the present invention is from about 1 mg/kg, up to about 3, about 6or about 12 mg/kg of body weight of the patient.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired.

Usually a dosage of active ingredient can be about 0.1 to 100 milligramsper kilogram of body weight. Ordinarily 0.1 to 50, and, preferably, 0.1to 10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one proteinscaffold of the present invention about 0.1 to 100 mg/kg or any range,value or fraction thereof per day, on at least one of day 1-40, or,alternatively or additionally, at least one of week 1-52, or,alternatively or additionally, at least one of 1-20 years, or anycombination thereof, using single, infusion or repeated doses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.001 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the protein scaffold can be formulated asa solution, suspension, emulsion, particle, powder, or lyophilizedpowder in association, or separately provided, with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and about 1-10% humanserum albumin. Liposomes and nonaqueous vehicles, such as fixed oils,can also be used. The vehicle or lyophilized powder can containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one protein scaffold according to the present invention. Whilepulmonary administration is used in the following description, othermodes of administration can be used according to the present inventionwith suitable results. Protein scaffolds of the present invention can bedelivered in a carrier, as a solution, emulsion, colloid, or suspension,or as a dry powder, using any of a variety of devices and methodssuitable for administration by inhalation or other modes described herewithin or known in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols, such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent, such as aqueous solution, asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least oneprotein scaffold by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermalmeans. At least one protein scaffold composition can be prepared for usefor parenteral (subcutaneous, intramuscular or intravenous) or any otheradministration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms, such as, but not limited to, creams and suppositories;for buccal, or sublingual administration, such as, but not limited to,in the form of tablets or capsules; or intranasally, such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally, such as not limited to a gel, ointment,lotion, suspension or patch delivery system with chemical enhancers suchas dimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement;” Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathways,such as electroporation, or to increase the mobility of charged drugsthrough the skin, such as iontophoresis, or application of ultrasound,such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably, at least one protein scaffoldcomposition is delivered in a particle size effective for reaching thelower airways of the lung or sinuses. According to the invention, atleast one protein scaffold can be delivered by any of a variety ofinhalation or nasal devices known in the art for administration of atherapeutic agent by inhalation. These devices capable of depositingaerosolized formulations in the sinus cavity or alveoli of a patientinclude metered dose inhalers, nebulizers, dry powder generators,sprayers, and the like. Other devices suitable for directing thepulmonary or nasal administration of protein scaffolds are also known inthe art. All such devices can use formulations suitable for theadministration for the dispensing of protein scaffold in an aerosol.Such aerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles.

Metered dose inhalers like the Ventolin® metered dose inhaler, typicallyuse a propellent gas and require actuation during inspiration (See,e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like Turbuhaler™(Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura),devices marketed by Inhale Therapeutics, and the Spinhaler® powderinhaler (Fisons), use breath-actuation of a mixed powder (U.S. Pat. No.4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura,U.S. Pat. No. 5,458,135 Inhale, WO 94/06498 Fisons, entirelyincorporated herein by reference). Nebulizers like AERx™ Aradigm, theUltravent® nebulizer (Mallinckrodt), and the Acorn II® nebulizer(Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO97/22376), the above references entirely incorporated herein byreference, produce aerosols from solutions, while metered dose inhalers,dry powder inhalers, etc. generate small particle aerosols. Thesespecific examples of commercially available inhalation devices areintended to be a representative of specific devices suitable for thepractice of this invention, and are not intended as limiting the scopeof the invention.

Preferably, a composition comprising at least one protein scaffold isdelivered by a dry powder inhaler or a sprayer. There are severaldesirable features of an inhalation device for administering at leastone protein scaffold of the present invention. For example, delivery bythe inhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g., less than about 10 μm, preferably about 1-5 μm, forgood respirability.

Administration of Protein Scaffold Compositions as a Spray

A spray including protein scaffold composition can be produced byforcing a suspension or solution of at least one protein scaffoldthrough a nozzle under pressure.

The nozzle size and configuration, the applied pressure, and the liquidfeed rate can be chosen to achieve the desired output and particle size.An electrospray can be produced, for example, by an electric field inconnection with a capillary or nozzle feed. Advantageously, particles ofat least one protein scaffold composition delivered by a sprayer have aparticle size less than about 10 μm, preferably, in the range of about 1μm to about 5 μm, and, most preferably, about 2 μm to about 3 μm.

Formulations of at least one protein scaffold composition suitable foruse with a sprayer typically include protein scaffold composition in anaqueous solution at a concentration of about 0.1 mg to about 100 mg ofat least one protein scaffold composition per ml of solution or mg/gm,or any range, value, or fraction therein. The formulation can includeagents, such as an excipient, a buffer, an isotonicity agent, apreservative, a surfactant, and, preferably, zinc. The formulation canalso include an excipient or agent for stabilization of the proteinscaffold composition, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating proteinscaffold compositions include albumin, protamine, or the like. Typicalcarbohydrates useful in formulating protein scaffold compositionsinclude sucrose, mannitol, lactose, trehalose, glucose, or the like. Theprotein scaffold composition formulation can also include a surfactant,which can reduce or prevent surface-induced aggregation of the proteinscaffold composition caused by atomization of the solution in forming anaerosol. Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitol fatty acid esters. Amounts will generally range between 0.001and 14% by weight of the formulation. Especially preferred surfactantsfor purposes of this invention are polyoxyethylene sorbitan monooleate,polysorbate 80, polysorbate 20, or the like. Additional agents known inthe art for formulation of a protein, such as protein scaffolds, orspecified portions or variants, can also be included in the formulation.

Administration of Protein Scaffold Compositions by a Nebulizer

Protein scaffold compositions of the invention can be administered by anebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically,in a jet nebulizer, a compressed air source is used to create ahigh-velocity air jet through an orifice. As the gas expands beyond thenozzle, a low-pressure region is created, which draws a solution ofprotein scaffold composition through a capillary tube connected to aliquid reservoir. The liquid stream from the capillary tube is shearedinto unstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of protein scaffold composition either directly or through acoupling fluid, creating an aerosol including the protein scaffoldcomposition. Advantageously, particles of protein scaffold compositiondelivered by a nebulizer have a particle size less than about 10 μm,preferably, in the range of about 1 μm to about 5 μm, and, mostpreferably, about 2 μm to about 3 μm.

Formulations of at least one protein scaffold suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one protein scaffold per mlof solution. The formulation can include agents, such as an excipient, abuffer, an isotonicity agent, a preservative, a surfactant, and,preferably, zinc. The formulation can also include an excipient or agentfor stabilization of the at least one protein scaffold composition, suchas a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating at least one protein scaffoldcompositions include albumin, protamine, or the like. Typicalcarbohydrates useful in formulating at least one protein scaffoldinclude sucrose, mannitol, lactose, trehalose, glucose, or the like. Theat least one protein scaffold formulation can also include a surfactant,which can reduce or prevent surface-induced aggregation of the at leastone protein scaffold caused by atomization of the solution in forming anaerosol. Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbital fatty acid esters. Amounts will generally range between about0.001 and 4% by weight of the formulation. Especially preferredsurfactants for purposes of this invention are polyoxyethylene sorbitanmono-oleate, polysorbate 80, polysorbate 20, or the like. Additionalagents known in the art for formulation of a protein, such as proteinscaffold, can also be included in the formulation.

Administration of Protein Scaffold Compositions by a Metered DoseInhaler

In a metered dose inhaler (MDI), a propellant, at least one proteinscaffold, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably, about 1 μm to about 5 μm, and, most preferably, about 2 μmto about 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of protein scaffold composition produced byvarious methods known to those of skill in the art, includingjet-milling, spray drying, critical point condensation, or the like.Preferred metered dose inhalers include those manufactured by 3M orGlaxo and employing a hydrofluorocarbon propellant. Formulations of atleast one protein scaffold for use with a metered-dose inhaler devicewill generally include a finely divided powder containing at least oneprotein scaffold as a suspension in a non-aqueous medium, for example,suspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably, the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one protein scaffold as a suspension inthe propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some cases,solution aerosols are preferred using solvents, such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation. One of ordinary skill in the art willrecognize that the methods of the current invention can be achieved bypulmonary administration of at least one protein scaffold compositionvia devices not described herein.

Oral Formulations and Administration

Formulations for oral administration rely on the co-administration ofadjuvants (e.g., resorcinols and nonionic surfactants, such aspolyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) toincrease artificially the permeability of the intestinal walls, as wellas the co-administration of enzymatic inhibitors (e.g., pancreatictrypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) toinhibit enzymatic degradation. Formulations for delivery of hydrophilicagents including proteins and protein scaffolds and a combination of atleast two surfactants intended for oral, buccal, mucosal, nasal,pulmonary, vaginal transmembrane, or rectal administration are taught inU.S. Pat. No. 6,309,663. The active constituent compound of thesolid-type dosage form for oral administration can be mixed with atleast one additive, including sucrose, lactose, cellulose, mannitol,trehalose, raffinose, maltitol, dextran, starches, agar, arginates,chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin,collagen, casein, albumin, synthetic or semisynthetic polymer, andglyceride. These dosage forms can also contain other type(s) ofadditives, e.g., inactive diluting agent, lubricant, such as magnesiumstearate, paraben, preserving agent, such as sorbic acid, ascorbic acid,.alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder,thickener, buffering agent, sweetening agent, flavoring agent, perfumingagent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 and used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration

A formulation for orally administering a bioactive agent encapsulated inone or more biocompatible polymer or copolymer excipients, preferably, abiodegradable polymer or copolymer, affording microcapsules which due tothe proper size of the resultant microcapsules results in the agentreaching and being taken up by the folliculi lymphatic aggregati,otherwise known as the “Peyer's patch,” or “GALT” of the animal withoutloss of effectiveness due to the agent having passed through thegastrointestinal tract. Similar folliculi lymphatic aggregati can befound in the bronchei tubes (BALT) and the large intestine. Theabove-described tissues are referred to in general as mucosallyassociated lymphoreticular tissues (MALT). For absorption throughmucosal surfaces, compositions and methods of administering at least oneprotein scaffold include an emulsion comprising a plurality of submicronparticles, a mucoadhesive macromolecule, a bioactive peptide, and anaqueous continuous phase, which promotes absorption through mucosalsurfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat.No. 5,514,670). Mucous surfaces suitable for application of theemulsions of the present invention can include corneal, conjunctival,buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal,and rectal routes of administration. Formulations for vaginal or rectaladministration, e.g., suppositories, can contain as excipients, forexample, polyalkyleneglycols, vaseline, cocoa butter, and the like.Formulations for intranasal administration can be solid and contain asexcipients, for example, lactose or can be aqueous or oily solutions ofnasal drops. For buccal administration, excipients include sugars,calcium stearate, magnesium stearate, pregelinatined starch, and thelike (U.S. Pat. No. 5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the at least one protein scaffold isencapsulated in a delivery device, such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers, such as polyhydroxy acids, such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers, such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

Prolonged Administration and Formulations

It can be desirable to deliver the compounds of the present invention tothe subject over prolonged periods of time, for example, for periods ofone week to one year from a single administration. Various slow release,depot or implant dosage forms can be utilized. For example, a dosageform can contain a pharmaceutically acceptable non-toxic salt of thecompounds that has a low degree of solubility in body fluids, forexample, (a) an acid addition salt with a polybasic acid, such asphosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid,pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- ordi-sulfonic acids, polygalacturonic acid, and the like; (b) a salt witha polyvalent metal cation, such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, orwith an organic cation formed from e.g., N,N′-dibenzyl-ethylenediamineor ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinctannate salt. Additionally, the compounds of the present invention or,preferably, a relatively insoluble salt, such as those just described,can be formulated in a gel, for example, an aluminum monostearate gelwith, e.g., sesame oil, suitable for injection. Particularly preferredsalts are zinc salts, zinc tannate salts, pamoate salts, and the like.Another type of slow release depot formulation for injection wouldcontain the compound or salt dispersed for encapsulation in a slowdegrading, non-toxic, non-antigenic polymer, such as a polylacticacid/polyglycolic acid polymer for example as described in U.S. Pat. No.3,773,919. The compounds or, preferably, relatively insoluble salts,such as those described above, can also be formulated in cholesterolmatrix silastic pellets, particularly for use in animals. Additionalslow release, depot or implant formulations, e.g., gas or liquidliposomes, are known in the literature (U.S. Pat. No. 5,770,222 and“Sustained and Controlled Release Drug Delivery Systems”, J. R. Robinsoned., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Examples Example 1 Tencon Design

The third FN3 domain from human Tenascin (SEQ ID NO: 3) can be used asan alternative scaffold capable of being engineered to bind to specifictarget molecules via surface exposed loops structurally analogous toantibody complementarity determining regions (CDR). The meltingtemperature of this domain is 54° C. in PBS in its native form. In orderto produce a scaffold molecule with a similar structure and improvedphysical properties, such as an improved thermal stability, a consensussequence was designed based on an alignment of 15 FN3 domains from humanTenascin (SEQ ID NOS: 1-15).

Analysis of the multiple sequence alignment in Table 1 shows that these15 domains have sequence identities to each other ranging from 13 to80%, with an average sequence identity among pairs of 29%. A consensussequence (SEQ ID NO: 16) was designed by incorporating the mostconserved (frequent) amino acid at each position from the alignmentshown in Table 1. In pairwise alignments, the consensus sequence of thepresent invention (SEQ ID NO:16), designated Tencon, is identical to theFN3 domains from Tenascin at 34-59% of positions with an averagesequence identity of 43%.

Expression and Purification

The amino acid sequence of Tencon (SEQ ID NO: 16) was back translated,resulting in the DNA sequence shown in SEQ ID NO: 17. This sequence wasassembled by overlapping PCR, subcloned into a modified pET 15 vector,transformed into BL21Star(DE3) E. coli (Invitrogen) and plated onto LBagar plates containing 75 μg/mL carbenicillin. A single colony waspicked and grown overnight at 37° C. in 50 ml of TB media containing 2%glucose and 100 μg/mL carbenicillin. This culture was used to seed 500mL of autoinduction media (Overnight Express Instant TB media, Novagen)in a 2.5 L Ultra Yield flask (Thomson Instrument Company). The growthand expression was done using a dual program (3 hours at 37° C., 300rpm, followed by 16 hours at 30° C., 250 rpm) in an ATR Multitronshaking incubator.

The culture was harvested and centrifuged at 7000 rpm for 15 minutes ina JL8.1 rotor to pellet the cells. The cells were resuspended in 30 mlbuffer containing 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10%glycerol, 20 mM imidazole, 0.37 mg/mL lysozyme, 1× Complete Proteaseinhibitor (EDTA-free; Roche) and Benzonase (Sigma-Aldrich, 0.25 μl/mlfinal) and lysed with a Misonix XL2020 sonicator for 5 minutes on ice inpulse mode (5 seconds on, 30 seconds off). The insoluble material wasremoved by centrifugation at 17,000 rpm for 30 minutes in a JA-17 rotor.

The Tencon protein was purified from the soluble lysate in a 2-stepchromatographic process. First, the protein was captured by immobilizedmetal affinity chromatography, adding 2 mL Ni-NTA agarose beads (Qiagen)to the lysate and placing it on a rocking platform for 1 hour at 4° C.The resin was then packed into a Poly-Prep column (Bio-Rad) and washedwith 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 20 mMimidazole to remove the unbound material. The proteins were eluted fromthe resin with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glyceroland 500 mM imidazole. The fractions were analyzed by SDS-PAGE, both byCoomassie stain and by Western blot using an HRP-conjugated anti-Hisantibody (Immunology Consultants Laboratory). The desired fractions werepooled and dialyzed into PBS pH 7.4. As a second purification step theprotein was loaded onto a Superdex-75 HiLoad 16/60 column (GEHealthcare) equilibrated in PBS. The fractions were analyzed bySDS-PAGE, and the fractions containing Tencon were pooled andconcentrated using a Centriprep UltraCel YM-3 concentrator (Amicon).

Protein concentration was determined using a BioTek plate reader tomeasure the absorbance of the sample at 280 nm. The final preparationwas analyzed by Coomassie stain (FIG. 1), Western blot with anti-Hisantibody, and by HPLC-SEC using a G3000SW-XL column (TOSOH Biosciences)equilibrated in PBS. SDS-PAGE analysis shows that Tencon migratesbetween 6 and 14 kDa, in agreement with the expected mass of 10.7 kDafor the monomeric protein. A yield of >50 mg of pure Tencon protein perliter of culture was obtained.

Biophysical Characterization

The structure and stability of Tencon was characterized by circulardichroism spectroscopy and differential scanning calorimetryrespectively. CD measurements were made on an AVIV spectrometer at 20°C. in PBS and a concentration of 0.2 mg/mL. The spectrum in FIG. 2 showsa minimum at 218 nm, suggestive of β-sheet structure as expected for aprotein belonging to the FN3 family as designed. DSC data was obtainedby heating 0.5 mg/mL solutions of the 3^(rd) FN3 domain from Tenascin orTencon in PBS from 35° C. to 95° C. at a rate of 1° C./minute in anN-DSCII calorimeter (Applied Thermodynamics). A buffer only curve wassubtracted to produce the profiles shown in FIG. 3. From this data,melting temperatures of 54° C. and 78° C. were calculated for the 3^(rd)FN3 domain and Tencon, respectively, using CpCalc (AppliedThermodynamics) software. The folding and unfolding of both domains isreversible at these temperatures.

Immunogenicity Analysis

A computer program that models for immunogenicity to human of amino acidsequences was used to compare the predicted immunogenicity of amino acidsequences representing the 3^(rd) FN3 domain of human Tenascin, Tencon,and several therapeutic antibodies (shown in Table 2). Chimeric mAbs anda human mAb (adalimumab) analyzed with the program were followed byapplication of a tolerance threshold (removes 9-mer peptides with 100%identity to human germline encoded sequence). The tolerance thresholdwas not applied to Tenascin or Tencon. The tolerance threshold assumesbroad T cell tolerance to germline encoded mAb sequences and focusesanalyses on novel sequence primarily in CDRs and flanking domains.

These analyses predict a low immunogenic risk for both Tenascin andTencon based on the likelihood that a 9-mer peptide, derived from theanalyzed sequence will bind one or more HLA molecules. The score isweighted with respect to the prevalence of each HLA allele. The scoresfor the models were summed for each sequence to provide a single numberdescribing the overall PIR of each sequence (score sum). The resultsfrom this analysis are summarized in Table 2. Tenascin was shown to havethe lowest overall Score (11.9). Tencon, like Tenascin, scored primarilynon-binders and low predicted immunogenic risk agretopes (Score=13.2).The Tenascin and Tencon sequences scored favorably as compared to thetherapeutic antibodies.

Display of Tencon on M13 Phage by pIX fusion

The gene encoding the Tencon amino acid sequence was subcloned into thephagemid expression vector pPep9 by PCR and restriction digest cloning,resulting in the vector pTencon-pIX. This system expresses N-terminallyMyc-tagged Tencon as a C-terminal fusion to the N-terminus of the M13pIX protein (FIG. 4). The Lac promoter allows for lower levels ofexpression without IPTG and increased expression after the addition ofIPTG. The OmpA signal sequence was appended to the N-terminus of Tenconto promote efficient translocation to the periplasm. A short TSGGGGSlinker (SEQ ID NO:18) was constructed between Tencon and pIX to preventsteric interactions between these proteins.

For confirmation of display on the surface of the M13 phage particle,pTencon-pIX was transformed into XL1-Blue E. coli and a single colonywas used to innocualte a 5 mL LB culture supplemented with ampicillin.This culture was grown at 37° C. until reaching mid-log phase at whichpoint 6¹⁰ pfu of VCSM13 helper phage was added and the culture incubatedat 37° C. for 10 minutes without shaking followed by 50 minutes withshaking The helper phage rescued culture was then diluted into 50 mL of2YT media supplemented with ampicillin and kanamycin and grown at 37° C.with shaking until O.D.₆₀₀ reached 0.7, at which point IPTG was added toa final concentration of 1 mM and the temperature reduced to 30° C.After 16 hours, the culture was centrifuged at 4000×g for 20 minutes andthe supernatant collected and stored at 4° C. for analysis.

Binding of the phage particles to an anti-Myc antibody (Invitrogen) wasused to confirm the display of the Myc-Tencon construct on the M13 phagesurface. A Maxisorp plate was coated overnight at a concentration of 2.5μg/mL with α-Myc or an anti-αv antibody (negative control) and blockedwith SuperBlock T20 (Pierce). Two-fold serial dilutions of the phagemidculture supernatant described above were made in PBS and added to thewells of the coated plate. After 1 hour, the plate was washed with TBSTand a α-M13 HRP antibody was added to each well and washed with TBSTfollowing a 1-hour incubation. The Roche BD ELISA POD substrate wasadded and luminescence detected on a plate reader (Tecan). FIG. 5 showsthat the Myc-Tencon phage particles bind to the α-myc, but not theanti-αv antibody coated wells or the uncoated control wells of the platein a concentration dependent manner, confirming the specific display ofMyc-Tencon on the M13 phage particle.

An additional phagemid vector can be constructed to display Tencon andlibrary members (see Example 2) on M13 phage as fusions to coat proteinpIII. For this system, the gene for pIX is replaced with a gene encodinga truncated version of pIII (Bass et al. 1990). Additional changes ascompared to the system shown in FIG. 4 include the replacement of theOmpA signal sequence with the signal sequence for DsbA, as secretionusing this sequence has been shown to be beneficial for the display ofstable alternative scaffold molecules (Steiner et al. 2006).

Example 2 Generation of Tencon Libraries

Tencon variant libraries can be made by many different methods,depending on the desired complexity and the relative location ofmutations in the molecule. DNA synthesis methods are preferred to createmutations scattered throughout the Tencon gene. Restriction enzymecloning can also be used to recombine DNA fragments containing mutationsin different regions of the gene. Saturating mutagenesis in asmall-defined region, such as a single Tencon loop, can be introduced byusing a degenerate oligo-nucleotide and oligonucleotide directedmutagenesis (Kunkel et al. 1987).

A Tencon library, library FG7, designed to replace the FG loop with 7random amino acids using oligonucleotide directed mutagenesis wasconstructed. An oligonucleotide (TconFG7-For-5′pho) was synthesized tohave a 21 base pair (bp) degenerate sequence of NNS at the positionsencoding the FG loop and two flanking 20-27 by nucleotide sequences ofcomplementarity to the Tencon coding sequence. In this design, alltwenty amino acids are capable of being represented in the FG loop. Thecalculated diversity at nucleotide level is 1.3×10⁹.

TconFG7-For5′pho: (SEQ ID NO: 19)GAATACACCGTTTCTATCTACGGTGTTNNSNNSNNSNNSNNSNNSNNSCC GCTGTCTGCGGAATTCAC

The template for oligonucleotide directed mutagenesis,pDsbA-Tencon-Asc-loop-Myc-pIII, was constructed by replacing the TenconF:G loop encoding sequence with a stem loop sequence containing a AscIrestriction site. This system allows the elimination of backgroundtemplate DNA after mutagenesis by digesting the resulting DNA with AscIprior to transformation. To purify a single-stranded DNA template formutagenesis, a single colony of E. coli CJ236 harboringpDsbA-Tencon-Asc-loop-Myc-pIII, was picked into 5 mL of 2YT growthmedium with carbenicillin (50 ug/ml final concentration) andChloramphenicol (10 ug/ml). After 6 hours, VCSM13 helper phage was addedto a final concentration of 10¹⁰ pfu/ml and incubated without shakingfor 10 minutes before being transferred to 150 mL of 2YT withcarbenicillin (10 ug/ml) and uridine (0.25 ug/ml) and incubated at 37°C. with shaking at 200 rpm overnight. The cells were pelleted bycentrifugation and the supernatant collected and the phage pelleted withPEG NaCl. Single strand DNA was purified from this pellet using aQIAprep Spin M13 kit (Qiagen) according to the manufacturerinstructions.

To anneal the degenerate oligonucleotide to the template, 5 μg oftemplate DNA was combined with oligo TconFG7-For-5-pho at a molar ratioof 10:1 in Tris-HCl (50 mM, pH7.5) and MgCl2 (10 mM) and incubated at90° C. for 2 minutes, 60° C. for 3 minutes, and 20° C. for 5 minutes.After the annealing reaction, ATP (10 mM), dNTPs (25 mM each), DTT (100mM), T4 ligase (7 units), and T7 DNA polymerase (10 units) were added tothe reaction mixture and incubated at 14° C. for 6 hours followed by 20°C. for 12 hours. The resulting DNA was purified using a PCR purificationkit (Qiagen) and recovered in 100 μL of water. The library DNA wasdigested with 10 units of AscI for 4 hours and then purified again withQiagen PCR purification kit. The final library DNA was recovered in 50μL of water. The resulting double stranded DNA product was thentransformed into into E. coli MC1061F′ by electroporation.

The transformants were collected in 20 mL SOC medium and allowed torecover for 1 hour at 37° C. At the end of the recovery, an aliquot ofthe transformation was serial diluted and plated on Carbenicillin (100ug/ml) plates containing 1% glucose to assess the total transformantnumber. The remaining SOC culture was then used to inoculate 1 L of 2×YT medium with Carbinicillin and 1% glucose and grown until OD₆₀₀reached 0.6. 100 mL of this culture was inoculated with M13 helper phageto 10¹⁰/mL and incubated at 37° C. before centrifugation. The resultingcell pellet was resuspended in 500 mL fresh 2×YT medium containingCarbenicillin (100 ug/mL) and Kanamycin (35 ug/mL) and grown at 30° C.overnight before centrifugation. Phage particles were precipitated bythe addition of PEG/NaCl and stored at −80° C.

A second library, BC6/FG7, was designed to introduce diversity withinthe B:C and F:G loops of Tencon simultaneously. In order to do so, twooligonucleotides, Tc-BC6-For-5′phos and POP149 were synthesized. Theforward oligo was phosphorylated and contained 18 bases of NNS codon ateach position encoding the B:C loop, while the reverse oligo wasbiotinylated at the 5′ end and contained 21 bases of NNS codon at eachposition encoding the F:G loop. Both oligonucleotides are flanked by two18 by nucleotide sequences identical to the region preceding andfollowing the region to be mutagenized (see below for primer detail).

Tc-BC6-For-5′phos: (SEQ ID NO: 20)GACTCTCTGCGTCTGTCTTGGNNSNNSNNSNNSNNSNNSTTCGACTCTTT CCTGATCCAGTACC POP2149: (SEQ ID NO: 21) GTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGTG

To construct the library, sixteen 100 μL PCR reactions were performedusing t oligos Tc-CB6-For4′phos and POP2149 to amplify the Tencon DNAtemplate, introducing NNS codons into the B:C and F:G loopssimultaneously in the process. The double-stranded PCR product was mixedwith magnetic streptavidin beads (Dynal) in B&W buffer (10 mM Tris-HCl,pH7.5, 1 mM EDTA, 2M NaCl, 0.1% Tween-20) and incubated for 20 minutes,pulled down with a magnet and washed with B&W buffer twice. The forwardstrand was eluted from the beads with 300 μL of 150 mM NaOH. This“megaprimer,” a mixture of a long primers with more than 8×10¹⁶ intheoretical diversity, was used to anneal to a single strand librarytemplate. Library construction was carried out as described above forthe FG7 library.

Example 3—Selection of TNFα Binders

In order to perform selections of Tencon library members that bind toTNFα, recombinant TNFα was biotinylated using sulfo-NHS-LC-Biotin(Pierce) before dialyzing into PBS. For selections, 200 μL of phagedisplaying libraries FG7 or BC6/FG7 were blocked with 200 μL ofchemiblocker before the addition of biotinylated TNFα at concentrationsof 500 nM (round 1) or 100 nM (rounds 2 and 3). Bound phages wererecovered by Neutravidin magnetic beads (Seradyne) in round 1 orstreptavidin magnetic beads (Promega) in rounds 2 and 3. Unbound phageswere washed from the beads using 5-10 washes with 1 mL of Tris bufferedsaline with tween (TBST) followed by 2 1 mL washes with Tris bufferedsaline (TBS). Bound phages were eluted from the beads by the addition ofmid-log phase E. coli MC1061F′. Infected cells were plated on LB agarplates supplemented with carbenicillin and glucose. The next day, cellswere scraped from the plate and grown to mid-log phase before rescuewith VCSM13 helper phage and grown overnight. Phage particles isolatedby PEG/NaCl precipitation and used for the next round of selections.

After 3 rounds of panning against TNFα, the output was subcloned into apET27 vector (Novagen) modified to include a ligase independent cloningsite by amplifying the Tencon gene by PCR. This PCR product was annealedto the vector and transformed into BL21-GOLD(DE3) cells (Stratagene).Individual colonies were picked into 1 mL cultures in 96 deep wellplates (Corning) and grown to saturation overnight at 37° C. The nextday, 50 μL of the overnight culture was used to inoculate a fresh 1 mLculture. Cultures were grown at 37° C. for 2 hours before adding IPTG to1 mM and reducing the temperature to 30° C. Cells were harvested bycentrifugation 16 hours after induction and lysed with 100 μL ofBugBuster (Novagen). The resulting lysates were clarified bycentriguation and used to test for binding to TNFα by ELISA.

Maxisorp plates (Nunc) were coated with 0.5 μg of streptavidin, washedwith TBST, and bound to 100 ng of biotinylated TNFα or 200 ng ofbiotinylated human serum albumin before being blocked with StartingBlock (Thermo Scientific). Clarified lysates diluted 1:1 in StartingBlock were added to the plates and allowed to bind for 1 hour beforebeing washed with TBST. Detection of bound Tencon was accomplished usingan anti-HIS-HRP antibody (Qiagen). Results of the ELISA are shown inFIG. 7. Constructs that bound biotinylated TNFα more than 10-fold overbiotinylated HSA as judged by ELISA signal were sequened. Aftercompletion of several selection experiments, 28 unique binding sequencesfrom library FG7 and 51 unique sequences from library BC6/FG7 wereobtained; Table 4 shows representative sequences of TNFα binders.Library BC6/FG7 produced more unique sequences but a lower overall hitrate and lower ELISA signals than those from library FG7; Table 4 showsrepresentative sequences of TNFα binders in which the B:C and/or F:Gloops are shown to the extent they are different than those of SEQ IDNO:16. Also shown in Table 4 are numerous mutations in other regions ofthe scaffold.

The Tencon protein designed, expressed, and purified here has a thermalstability improved by 26° C. with respect to that of the 3^(rd) FN3domain from human Tenascin, which has been used as an alternativescaffold molecule. Based on this stability increase, this scaffoldmolecule is likely to be more amenable to amino acid substitution andeasier to manufacture. Mutations that decrease protein stability arelikely to be better tolerated in the context of a more stable scaffoldand thus a scaffold with enhanced stability is likely to yield morefunctional, well folded binders from a library of scaffold variants. Asthis novel protein is not a protein encoded by the human genome, it mayalso provide less risk toward the generation of an immune response thanthe risk against, for example, native human Tenascin when used as atherapeutic (essentially, less risk than with a therapeutic based on thewild type domain).

For the purposes of this invention, 70-100% amino acid or nucleotidesequence identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) is determined using a suitable computeralgorithm, as known in the art.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

TABLE 1 (1) 1       .10       .20       .30       .40       .50    ................................................... 1 (1)---SPPKDLVVTEVTEETVNLAWDN-EMRVTEYLVVYTPTH--EGGLEMQF 2 (1)TYLPAPEGLKFKSIKETSVEVEWDPLDIAFETWEIIFRNMN-KEDEGEITK 3 (1)---DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKD--VPGDRTTI 4 (1)TGLDAPRNLRRVSQTDNSITLEWRNGKAAIDSYRIKYAPISGGDHAEVDVP 5 (1)---DTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPT----GQNVGV 6 (1)-QAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEAN--KVEAARNL 7 (1)-ETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEAD--TVEAAQNL 8 (1)-EVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEAD--QVEEAHNL 9 (1)-DLPQLGDLAVSEWGWDGLRLNWTAADNAYEHFVIQVQEVN--KVEAAQNL 10 (1)-KEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSN--RLLETVEY 11 (1)-ALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSG--KLLDPQEF 12 (1)-AEPEVDNLLVSDATPDGFRLSWTADEGVFDNFVLKIRDTK--KQSEPLEI 13 (1)---GSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITG---GTPSMV 14 (1)---DGPSGLVTANITDSEALARWQPAIATVDSYVISYTGEK----VPEITR 15 (1)---DSPRDLTATEVQSETALLTWRPPRASVTGYLLVYESVD----GTVEKV       .60       .70       .80       .90      100    ................................................. 1 (1)RVPGDQTSTIIQELEPGVEYFIRVFAILENKKSIPVSARVAT------- 2 (1)SLRRPETSYRQTGLAPGQEYEISLHIVKNNTRGPGLKRVTTTRLD---- 3 (1)DLTEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT------- 4 (1)KSQQATTKTTLTGLRPGTEYGIGVSAVKEDKESNPATINAATELDTPKD 5 (1)QLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPAKSKPARVK----- 6 (1)TVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGE----- 7 (1)TVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTE------ 8 (1)TVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTE------ 9 (1)TLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPE-- 10 (1)NISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTE------ 11 (1)TLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTE------ 12 (1)TLLAPERTRDLTGLREATEYEIELYGISKGRRSQTVSAIATTAM----- 13 (1)TVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAL----- 14 (1)TVSGNTVEYALTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDL----- 15 (1)IVGPDTTSYSLADLSPSTHYTAKIQALNGPLRSNMIQTIFTTIGL----

TABLE 2 1^(st) Score 2^(nd) Score Score sum Score sum SequenceDescription sum sum (chain) (molecule) Tenascin Alt. Scaff. 6.01 5.8511.86 11.86 Tencon Alt. Scaff. 5.83 7.37 13.20 13.20 adalimumab VhHumanized mAb 9.45 8.06 17.50 45.42 Vl 15.29 12.63 27.92 cetuximab VhChimeric mAb 17.63 16.89 34.52 64.44 Vl 14.45 15.47 29.92 Rituximab VhChimeric mAb 16.57 14.38 30.96 61.65 Vl 16.63 14.06 30.69 basiliximab VhChimeric mAb 16.48 13.40 29.89 58.98 Vl 16.05 13.05 29.09

Sequences:

SEQ ID No. 1: sppkdlvvtevteetvnlawdnemrvteylvvytpthegglemqfrvpgdqtstiiqelepgveyfirvfailenkksipvsarvat SEQ ID No. 2:tylpapeglkfksIketsvevewdpldiafetweiifrnmnkedegeitkslrrpetsyrqtglapgqeyeislhivknntrgpglkrvtttrld SEQ ID No. 3:dapsqievkdvtdttalitwfkplaeidgieltygikdvpgdrttidltedenqysignlkpdteyevslisrrgdmssnpaketftt SEQ ID No. 4tgldaprnlrrvsqtdnsitlewrngkaaidsyrikyapisggdhaevdvpksqqattkttltglrpgteygigvsavkedkesnpatinaateldtpkd SEQ ID No. 5dtpkdlqvsetaetsltllwktplakfdryrlnyslptgqwvgvqlprnttsyvlrglepgqeynvlltaekgrhkskpakskparvk SEQ ID No. 6qapelenltvtevgwdglrlnwtaadqayehfiiqvqeankveaarnltvpgslravdipglkaatpytvsiygviqgyrtpvlsaeastge SEQ ID No. 7etpnlgevvvaevgwdalklnwtapegayeyffiqvqeadtveaaqnltvpgglrstdlpglkaathytitirgvtqdfsttplsvevlte SEQ ID No. 8evpdmgnltvtevswdalrlnwttpdgtydqftiqvqeadqveeahnltvpgslrsmeipglragtpytvtlhgevrghstrplavevvte SEQ ID No. 9dlpqlgdlavsevgwdglrlnwtaadnayehfviqvqevnkveaaqnltlpgslravdipgleaatpyrvsiygvirgyrtpvlsaeastakepe SEQ ID No. 10kepeignlnvsditpesfnlswmatdgifetftieiidsnrlletveynisgaertahisglppstdfivylsglapsirtktisatatte SEQ ID No. 11alpllenltisdinpygftvswmasenafdsflvtvvdsgklldpqeftlsgtqrklelrglitgigyevmvsgftqghqtkplraeivte SEQ ID No. 12aepevdnllvsdatpdgfrlswtadegvfdnfvlkirdtkkqsepleitllapertrdltglreateyeielygiskgrrsqtvsaiattam SEQ ID No. 13gspkevifsditensatvswraptaqvesfrityvpitggtpsmvtvdgtktqtrlvklipgveylvsiiamkgfeesepvsgsfttal SEQ ID No. 14dgpsglvtanitdsealarwqpaiatvdsyvisytgekvpeitrtvsgntveyaltdlepateytlrifaekgpqksstitakfttdl SEQ ID No. 15dsprdltatevqsetalltwrpprasvtgyllvyesvdgtvkevivgpdttsysladlspsthytakiqalngplrsnmiqtifttigl SEQ ID No. 16LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT SEQ ID No. 17ctgccggcgccgaaaaacctggttgtttctgaagttaccgaagactctctgcgtctgtcttggaccgcgccggacgcggcgttcgactctttcctgatccagtaccaggaatctgaaaaagttggtgaagcgatcaacctgaccgttccgggttctgaacgttcttacgacctgaccggtctgaaaccgggtaccgaatacaccgtttctatctacggtgttaaaggtggtcaccgttctaacccgctgt ctgcggaattcaccaccTencon Sequence showing loops (SEQ ID NO: 16)              A-B loop  B-Cloop        C-D loop 1-LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEA     D-E loop  E-F loop       F-G loopINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT-89

TABLE 3 Loons of Tencon Loop Residues of SEQ ID NO: 16 Amino AcidSequence A-B 13-16 TEDS B-C 22-28 TAPDAAF C-D 38-43 SEKVGE D-E 51-54GSER E-F 60-64 GLKPG F-G 75-81 KGGHRSN

TABLE 4 Scaffolds binding to TNFα B:C Loop F:G Loop Clone Residues 22-28Residues 75-81 Scaffold No. (SEQ ID NO) (SEQ ID NO) Mutations 1 GGLTYFH(78) 2 GGLSDIH (79) 3 EGLGVIH (80) 4 GGLGVIH (81) 5 GGLSVIH (82) 6GGLTVIH (83) 7 GGLTYIH (84) 8 GGLTYLH (85) 9 GGLSYFH (86) 10 GGLTDIH(87) 11 GGLSSIH (88) 12 GGLRVIH (89) 13 GALSVIH (90) 14 GGLSFLH (91) 15ITHMRDM (92) 16 KTRYCF (22) ANLFYLR (93) 17 SSSHGH (23) GALSVIH (90) 18ESRQAY (24) GNLTYLH (94) 19 NKLDSG (25) YNSTELG (95) 20 ESQASY (26)GQLAVIH (96) 21 NAPLLW (27) GNLSVLQ (97) 22 DPWYYY (28) TRNYVLT (98)P50S 23 TLYCRY (29) TPPCGYA (99) 24 NCTTTF (30) CWSYYMS (100) 25 QCSVCR(31) GGLTVLQ (101) 26 DSWYPS (32) RPNFFMG (102) 27 CLAVSP (33) CTTVTYH(103) 28 ECNPCR (34) GSLTILQ (104) 29 SCSFEC (35) DRRRPMR (105) R18P 30SAWSFY (36) GRNYTFR (106) 31 LQHCAS (37) GQLAVIH (96) 32 ESHGSY (38)GQLAVIR (107) 33 ETHDTN (39) GQLDDNL (108) 34 GGLTVLH (109) 35 GGLAYLH(110) 36 GGLSVVH (111) 37 ESRQTY (40) GNLTYLH (94) 38 SAWSFY (36)GNLTYLH (94) 39 ESRQAY (41) GKNYTFR (112) 40 SAWPFY (42) GKNYTFR (112)41 SAWSFY (36) GKNYTFR (112) 42 SAWSFY (36) GNNYVFK (113) 43 ESRASY (43)GQLAVIH (96) 44 TAKYWL (44) GQLAVIH (96) 45 TAKYWL (44) GSNYMFH (114) 46HPWWFL (45) GSNYMFH (114) 47 HPWWFL (45) GRNYLMS (115) 48 TAKYWL (44)GRNYLMS (115) 49 IQPRFY (46) HWGHNLT (116) 50 RQRYMV (47) LIAMLVS (117)51 RFPAFV (48) PTSCAYN (118) 52 CMRNPI (49) NLLRDSH (119) 53 TAKYWL (50)RPIVLPN (120) 54 EMKFLH (51) SSNYTFR (121) 55 REKRLR (52) VPTSALS (122)56 CGYGKM (53) FILPKLF (123) 57 CDLWSS (54) KYHYPYP (124) E66K 58 FIFAIM(55) LRLELRK (125) 59 YYRIIE (56) SMNLLSP (126) 60 PNSAMF (57) HSYGETL(127) 61 YSILEA (58) YCAPPMK (128) 62 KVCTAY (59) GMNSFSP (129) 63TEGKEG (60) SPWLARR (130) 64 MDIDHS (61) SLGYTTK (131) 65 GSMNEH (62)ENDKSPM (132) 66 SFSHTV (63) DESIPIS (133) 67 RMRTTE (64) QSADVFT (134)68 WHGMIRP (135) 69 VLSVLQR (136) 70 EWRVQVA (137) 71 GGLRYLH (138) 72MLKSFAS (139) 73 PWRYGLH (140) 74 GGLSYLH (141) 75 MTLTGID (142) 76GGWPVRV (143) 77 LDLYLRT (144) 78 RLTALNA (145) 79 GIPRWVR (146) 80RVSEEYG (147) 81 VLSLWMI (148) 82 WMVNKVL (149) 83 DVTEVYM (150) 84SVRMSSV (151) 85 LWWTLRK (152) 86 WAHLLRD (153) 87 LLTSE*S (154) 88*SGMHWG (155) 89 LRWYSV (65) SKFGVEG (156) 90 LRWYSV (65) TRFGVEQ (157)S30F 91 GTSTRR (66) TRFGVEQ (157) T48I 92 NFANVM (67) TRFGVEQ (157)S30F, V69L 93 NFANVM (67) TRFGVEQ (157) S30F 94 WKVGSV (68) LHHLTLH(158) 95 KASGRK (69) PSIASII (159) 96 LVLGAM (70) KTPHSRK (160) 97GRTFDW (71) KGRRHTK (161) N46D 98 FYLNFE (72) YRSKYTL (162) 99 NFANVM(73) MGTHLTL (163) S30F 100 TGQQSR (74) NSRTEIL (164) 101 LVLGAM (70)MLKCPAR (165) 102 GSRREV (75) FTSPMAT (166) 103 SRWKIG (76) TMPDCMT(167) 104 SVLIWQ (77) FDGYQCY (168)

1. An isolated protein scaffold based on a fibronectin type III (FN3)domain comprising an amino acid sequence derived from a consensussequence of the Tenascin FN3 domains, wherein protein scaffold iscapable of binding to human TNFα.
 2. The protein scaffold of claim 1,comprising 7 strands and 6 loops between the strands.
 3. An isolatedprotein scaffold based on a fibronectin type III (FN3) domain comprisingan amino acid sequence derived from a consensus sequence of the TenascinFN3 domains having the amino acid sequence of any one of the B:C or F:Gloops of SEQ ID NOS:22-168, wherein the protein scaffold forms loops ator about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 and iscapable of binding to human TNFα.
 4. An isolated protein scaffold basedon a fibronectin type III (FN3) domain comprising: a backbone portionhaving an amino acid sequence identical to SEQ ID NO:16 at residues1-21, 29-74, and 82-89; a B:C loop portion between residues 21 and 29 ofSEQ ID NO:16 having at least 75% identity to any one of SEQ IDNOS:22-77; and an F:G loop portion between residues 74 and 82 of SEQ IDNO:16 having at least 75% identity to any one of SEQ ID NOS:78-168,wherein the protein scaffold is capable of binding to human TNFα.
 5. Theprotein scaffold of claim 4, comprising: a backbone portion having anamino acid sequence identical to SEQ ID NO:16 at residues 1-21, 29-74,and 82-89; a B:C loop portion between residues 21 and 29 of SEQ ID NO:16comprising any one of SEQ ID NOS:22-77; and an F:G loop portion betweenresidues 74 and 82 of SEQ ID NO:16 comprising any one of SEQ IDNOS:78-168, wherein the protein scaffold is capable of binding to humanTNFα.
 6. The protein scaffold of claim 5, wherein said scaffold binds atarget with at least one affinity selected from a K_(D) of less than orequal to 10⁻⁹M, less than or equal to 10⁻¹⁰M, less than or equal to10⁻¹¹M, less than or equal to 10⁻¹²M, less than or equal to 10⁻¹³M, lessthan or equal to 10⁻¹⁴M, and less than or equal to 10⁻¹⁵M, as determinedby surface plasmon resonance or the Kinexa method.
 7. The proteinscaffold of claim 5, wherein the loops form sites for binding totargets.
 8. A method of constructing a library of a protein scaffoldbased on a fibronectin type III (FN3) domain capable of binding to humanTNFα, comprising the steps of: providing the protein scaffold of claim5; and introducing diversity into copies of the protein scaffold to formthe protein scaffold library.
 9. The method of claim 8, wherein theintroducing diversity step comprises mutating at least one loop regionselected from the group consisting of the residues at or about residues13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of the protein scaffoldamino acid sequence.
 10. A library produced by the method of claim 9.11. A method of generating a protein scaffold capable of binding tohuman TNFα with a predefined binding affinity, comprising contacting thelibrary of claim 10 with at least a portion of human TNFα protein andisolating a protein scaffold binding to human TNFα with the predefinedaffinity.
 12. The method of claim 11, wherein the isolating stepcomprising isolating scaffold molecules binding to human TNFα andtesting the isolated scaffold molecules for binding affinity to humanTNFα.
 13. The method of claim 12, wherein the affinity is a K_(D) lessthan or equal to about 10⁻⁷M.
 14. An isolated nucleic acid moleculeencoding the protein scaffold of claim
 5. 15. An isolated nucleic acidvector comprising the isolated nucleic acid molecule according to claim14.
 16. A prokaryotic or eukaryotic host cell comprising the isolatednucleic acid molecule according to claim
 14. 17. The host cell accordingto claim 16, wherein said host cell is at least one selected from E.coli BL21Star(DE3), other E. Coli cell, yeast, COS-1, COS-7, HEK293,BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. 18.A composition comprising a protein scaffold according to any of claims1-5 and at least one pharmaceutically acceptable carrier or diluent. 19.A composition according to claim 18, further comprising at least onecompound or polypeptide selected from a detectable label or reporter, ananti-infective drug, a cardiovascular (CV) system drug, a centralnervous system (CNS) drug, an autonomic nervous system (ANS) drug, arespiratory tract drug, a gastrointestinal (GI) tract drug, a hormonaldrug, a drug for fluid or electrolyte balance, a hematologic drug, anantineoplastic, an immunomodulation drug, an opthalmic, otic or nasaldrug, a topical drug, a nutritional drug, a cytokine, and a cytokineantagonist.
 20. A medical device, comprising a protein scaffoldaccording to any of claims 1-5, wherein said device is suitable forcontacting or administering said protein scaffold by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.21. An article of manufacture for human pharmaceutical or diagnosticuse, comprising packaging material and a container comprising a solutionor a lyophilized form of the protein scaffold of claim
 5. 22. Thearticle of manufacture of claim 21, wherein said container is acomponent of a parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracerebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermaldelivery device or system.
 23. Any invention described herein.